Leica LCS Manual PDF

Leica Confocal Software
LCS
User Manual
Table of Contents
Table of Contents ....................................................................................................................................2
Legal Notes .............................................................................................................................................5
Starting up the system.............................................................................................................................7
TM
Starting the Windows NT Operating System....................................................................................7
Using a Mouse .................................................................................................................................7
The Windows NT Interface...............................................................................................................8
The Start Menu.................................................................................................................................9
Starting a Program ...........................................................................................................................9
The Taskbar ...................................................................................................................................10
Setting the Time and Date .............................................................................................................10
Getting Help ...................................................................................................................................11
Shut Down Windows NT ................................................................................................................11
Setting Up Users Under WinNT .........................................................................................................12
The Leica Confocal Software: Overview ...............................................................................................13
Starting the Software......................................................................................................................13
The Experiment Software Concept ................................................................................................14
The Basic Structure of the User Interface......................................................................................14
Opening Data Records...................................................................................................................15
Saving Images................................................................................................................................15
Data Organization by Grouping Experiments ................................................................................16
Compiling Experiments ..................................................................................................................16
Menu Functions .................................................................................................................................16
Keyboard Shortcuts ...........................................................................................................................19
LCS Software Functions........................................................................................................................19
Introduction to the Leica Confocal Software Help..........................................................................19
Opening the Context-Sensitive Help ..............................................................................................20
Documentation Conventions ..........................................................................................................21
Data Recording ..................................................................................................................................22
Setting the Beam Path ...................................................................................................................22
Selecting an Objective ...................................................................................................................24
Setting the Detectors......................................................................................................................25
The Electronic Zoom ......................................................................................................................26
Enlarged Recording of a Frame .....................................................................................................28
Setting the Detection Pinhole.........................................................................................................29
Selecting a Scan Format................................................................................................................30
Selecting a Scan Mode ..................................................................................................................31
Selecting a Scan Speed.................................................................................................................32
Setting the Z/Y-Position .................................................................................................................33
Configuring a Time Series..............................................................................................................33
Starting a Single Scan....................................................................................................................34
Starting a Continuous Scan ...........................................................................................................35
Series Scan Overview Dialog Window...........................................................................................36
Defining the Begin Point for a Spatial Series .................................................................................37
Defining the Begin Point for a Wavelength Series .........................................................................37
Defining the End Point for a Spatial Series ....................................................................................38
Defining the End Point for a Wavelength Series............................................................................39
Defining the Number of Spatial Sections .......................................................................................40
Setting the Number of Wavelength Steps ......................................................................................41
Starting a Series Scan ...................................................................................................................41
Selecting a unidirectional or bi-directional scan.............................................................................43
Adjusting phase..............................................................................................................................44
Turning the Scan Field ...................................................................................................................44
Recording an Image of a Line Using the Averaging Method .........................................................45
Applying the Parameter Setting of an Experiment .........................................................................45
Recording an Image Using Burst Mode .........................................................................................46
Recording an image using the Averaging Method .........................................................................46
Image Recording With a Digital Resolution of 8 Bits or 12 Bits .....................................................47
Setting the UV Lens Wheel ............................................................................................................48
Data Viewing......................................................................................................................................48
The Viewer Window .......................................................................................................................48
Viewer Options ...............................................................................................................................51
Viewer Options Dialog Window, 3D Icon .......................................................................................51
Viewer Options Dialog Window, Display Icon ................................................................................52
Viewer Options Dialog Window, Charts Icon .................................................................................53
Viewer Options Dialog Window, Online Measure Icon ..................................................................55
Viewer Options Dialog Window, Projections Icon ..........................................................................56
Viewer Options Dialog Window, Overlay Icon ...............................................................................57
Viewer Options Dialog Window, Scan Progress Icon ....................................................................58
Viewer Options Dialog Window, Surface View Icon.......................................................................58
Viewer Options Dialog Window, Surface Measure Icon ................................................................60
Viewer Options Dialog Window, Surface Calculation Icon ............................................................61
Image Tool .....................................................................................................................................62
Image Tool Dialog Window ............................................................................................................62
Image Tool Dialog Window, 3D Button (optional) ..........................................................................64
Image Tool Dialog Window, Basic Button ......................................................................................71
Image Tool Dialog Window, Amplitude Button...............................................................................77
Viewing detection channels............................................................................................................83
Viewing Detection Channel 1-8......................................................................................................83
Zooming Image(s) in the Viewer Window ......................................................................................84
Selecting Color Maps (LUT)...........................................................................................................84
Viewing the Single image...............................................................................................................86
Viewing a Multiple image ...............................................................................................................87
Viewing an Overlay Image .............................................................................................................87
Viewing Image Series ........................................................................................................................88
Viewing the First Image of a Series ...............................................................................................88
Display the Next Image in a Series ................................................................................................88
Displaying the Previous Image in a Series ....................................................................................89
Viewing the Last Image of a Series................................................................................................89
Displaying an Image From a Series ...............................................................................................90
Starting and Ending a Film.............................................................................................................90
Viewing the Series image...............................................................................................................91
Projections .........................................................................................................................................92
Principles and Types of Projections ...............................................................................................92
Projecting an Image Stack with an Invariable Projection Axis .......................................................95
Projecting an Image Stack with a Variable Projection Axis (optional) ...........................................96
Maximum Projection of an Image Stack with Invariable Projection Axis .......................................97
Maximum Projection of an Image Stack with Invariable Projection Axis .......................................98
Average Projection of an Image Stack With Variable Projection Axis (optional) .........................100
Average Value Projection of an Image Stack with Invariable Projection Axis .............................101
Transparent Projection of an Image Stack with Invariable Projection Axis..................................102
Transparent Projection of an Image Stack with Variable Projection Axis (optional)....................103
Creating SFP projections of an image stack (optional)................................................................104
Creating a Topographical image ..................................................................................................105
Viewing the Original image ..........................................................................................................106
Creating 3D Views ...........................................................................................................................106
Creating the 3D View ...................................................................................................................106
Rotating the 3D View ...................................................................................................................107
Moving the 3D View .....................................................................................................................108
Zooming the 3D View...................................................................................................................108
Measuring and Analysis Functions......................................................................................................109
Calculating a Histogram ...............................................................................................................109
Measuring a Profile Within a Region of Interest (ROI).................................................................110
Measuring a Profile Along a Line Segment..................................................................................112
Measuring Surfaces and Volumes ...............................................................................................113
Copying Quantification Graphs to the Annotation Sheet .............................................................115
Printing Quantification Graphs .....................................................................................................115
Exporting Quantification Data ......................................................................................................116
Defining the Region of Interest (ROI) as an Ellipse .....................................................................116
Defining the Region of Interest (ROI) as a Polygon.....................................................................117
Defining the Region of Interest (ROI) as a Rectangle..................................................................118
Automatically Defining the Region of Interest (ROI) ....................................................................119
Selecting and Moving the Region of Interest (ROI) .....................................................................120
Moving and Rotating the Region of Interest (ROI).......................................................................121
Deleting Regions of Interest (ROIs) .............................................................................................122
Documenting Data...............................................................................................................................122
Creating an Annotation Sheet ......................................................................................................122
Copying an Image into the Annotation Sheet ..............................................................................123
Drawing a Line on the Annotation Sheet .....................................................................................124
Drawing a Rectangle on the Annotation Sheet ............................................................................124
Adding a Text Field to an Annotation Sheet ................................................................................125
Printing .........................................................................................................................................126
Data Handling......................................................................................................................................127
Opening a File ..............................................................................................................................127
Saving a File.................................................................................................................................127
Saving a File as............................................................................................................................128
Storing All Data ............................................................................................................................128
Creating an Experiment ...............................................................................................................129
User-specific Adaptation......................................................................................................................129
Saving the Viewer Window as a Template ..................................................................................129
Controlling Functions Using the Control Panel ............................................................................130
Optional Software packages ...............................................................................................................131
Materials ..........................................................................................................................................131
Image Tool dialog window, Materials button (optional)................................................................131
Measuring Roughness Along a Line Segment.............................................................................134
Measuring Roughness Within a Region of Interest (ROI)............................................................136
Glossary ..............................................................................................................................................138
Appendix..............................................................................................................................................141
LCS File Formats .............................................................................................................................144
Formats of User-specific and Device-dependent Data ................................................................144
Fixed Leica-specific File Formats.................................................................................................145
Index ....................................................................................................................................................157
Leica Microsystems Heidelberg GmbH
Legal Notes
Legal Notes
Version 2.0, August 01, 2001. Made in Germany.
© Copyright 2000/2001, Leica Microsystems Heidelberg GmbH. All rights
reserved.
No part of this publication may be reproduced or transmitted in any form

or by any means, electronic or mechanical, including photocopying,
recording, or storing in a retrieval system, or translating into any
language in any form without the express written permission of Leica
Microsystems Heidelberg GmbH.
DISCLOSURE
This document contains Leica Microsystems Heidelberg GmbH
proprietary data and is provided solely to its customers for their express
benefit of safe, efficient operation and maintenance of the product
described herein. Use or disclosure of Leica Microsystems Heidelberg
GmbH proprietary data for the purpose of manufacture or reproduction of
the item described herein, or any similar item, is prohibited, and delivery
of this document shall not constitute any license or implied authorization
to do so.
REVISIONS AND CHANGES

Leica Microsystems Heidelberg GmbH reserves the right to revise this
document and/or improve products described herein at any time without
notice or obligation. Information and specifications in this manual are
subject to change without notice.
WARRANTY
Leica Microsystems Heidelberg GmbH provides this publication "as is"
without warranty of any kind, either expressed or implied, including but
not limited to the implied warranties of merchantability or fitness for a
particular purpose. All reasonable precautions have been taken in the
preparation of this document, including both technical and non-technical
proofing. Leica Microsystems Heidelberg GmbH assumes no
responsibility for any errors or omissions. Leica Microsystems Heidelberg
GmbH shall not be responsible for any direct, incidental or consequential
damages arising from the use of any material contained in this document.
TRADEMARKS
Throughout this manual, trademarked names may be used. Rather than
including a trademark (™) symbol at every occurrence of a trademarked
name, we state that we are using the names only in an editorial fashion,
and to the benefit of the trademark owner, with no intention of
infringement.
SAFETY
This instrument is designed and manufactured to comply with applicable
performance standards for Class 3b laser products as defined by
USHHS, CDRH/FDA, OSHA and EN standards and regulations known to
be effective at the date of manufacture.
Every hazardous situation cannot be anticipated, therefore, the user must
exercise care, common sense, and observe all appropriate safety
precautions applicable to Class 3b lasers and high-voltage electrical
equipment during installation, operation and maintenance.
Deviation from published operating or maintenance procedures is not
recommended. Operation and maintenance procedure changes are
User Manual Leica LCS english Page 5 of 164

Art.No.: 15-9330-033 / Vers.: 01082001
Legal Notes
performed entirely at the user's risk.
SOFTWARE LICENSE
The software described in this document is furnished under a License
Agreement, which is included with the product. This agreement specifies
the permitted and prohibited uses of the product.

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LCS Software Functions
Starting up the system

Starting the Windows NTTM Operating
System
You don't have to start Windows NT™–it starts automatically when you
turn on your PC. You will first see a splash screen.
Next you have to log on to your computer. As you can see from the
instructions in the box, pressing the Ctrl, Alt and Delete keys at the
same time will log you on.
After pressing the Ctrl, Alt, and Delete keys, the Login information
dialog box appears.
Typing your password identifies you as a valid user for this computer.
The default user name for the Leica TCS SP2 system is "TCS_User."
A standard password was not set. It is recommended setting up a
separate user ID for each user (system administrator). This will create
individual directories that can be viewed only by the respective user.
Since the LCS software is based on the user administration of Windows
TM
NT , separate files are created for managing user-specific profiles of
the LCS software. For information about setting up users under Windows
TM
NT , please refer to the chapter "Setting Up Users under WinNT" in this
manual.
After logging on with your user ID, you may change your password by
pressing the keys Ctrl, Alt, and Delete at the same time.
Then click on Change Password. The Change Password dialog box
displays.
Type your current password in the Old Password field (passwords are
case sensitive, so be sure you use the right case). Then press the Tab
key. Pressing the Tab key moves the cursor to the next field.
Type your new password, then press the Tab key again. Confirm your
new password by re-entering it. This will eliminate any typing errors. This
is especially important since the characters you type appear as asterisks
on the screen.
Note
If you miss-type the confirmation password, you will see a warning

dialog. Try again!
Then click the OK button. Your new password will be in effect the next
time you log on.
Caution
Do not forget your password if you set one! Without the right password
you cannot access your computer anymore.
The Welcome dialog box is now displayed. Take a moment to read the
"Did you know..." tip and then click the Close button to begin using
Windows NT.
Using a Mouse
You need a mouse to work most efficiently in Windows NT. Here are the
mouse actions you need to know:
Point means to move the mouse pointer onto the specified item by
moving the mouse. The tip of the mouse pointer must touch the item.

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Click on an item means to move the pointer onto the specified item and
press and release the mouse button once. Unless specified otherwise
(i.e. right-click), use the left mouse button. Clicking usually selects an
item.
Double-click on an item means to move the pointer to the specified
item and press and release the left mouse button twice quickly. Double-
clicking usually activates an item.
Drag means to move the mouse pointer onto the item, hold down the
mouse button, and move the mouse while holding down the button.
Unless specified otherwise (i.e. right-drag), use the left mouse button.
The Windows NT Interface

The basic interface of Windows NT is called the "Desktop". It provides a
background for the items it contains.
The initial icons on the desktop allow the user to view and interact with
the system in a logical way.
The Windows NT screen contains many special elements and controls.
Here is a brief summary:
The background on which all the pictures and boxes rest, is the desktop.
The taskbar shows the windows and programs that are open. You can
switch between open windows and programs by clicking the name on the
taskbar.
The Start button opens a menu system from which you can start
programs. Click on the Start button, then click on your selection from
each menu that appears.
Some icons appear on your desktop. You can activate one by double
clicking on it.
We now take a brief tour of the items you see on the screen.
A standard desktop item is the My Computer icon. Double-clicking this
icon opens the My Computer window.
The 'My Computer' window gives you easy access to the major
components like hard and floppy disk drives of your computer system or
workstation. For example, by double-clicking the Hard disk [C:] icon you
can see the contents of your PC's hard drive. This allows the user to view
local resources as objects. The Windows NT Workstation 4 control panel
and print control/support are also accessible from 'My Computer.' If you
installed one of the additional applications such as 'Dial-Up Networking'
at installation time, it will also appear in the 'My Computer' window.
You can use the Control Panel icon in the 'My Computer' window to
view and change any system component. The Control Panel contains
numerous icons that allow you to control your system. The particular
icons that you see on your PC may be slightly different from those
illustrated, due to that fact that you may have different hardware installed,
and may or may not be connected to a network or modem. You may also
have different Windows NT Workstation 4 options installed.
Double-clicking the Network Neighborhood icon displays the Network
Neighborhood dialog box which gives you information about who and
what is connected to your workstation. It provides an easy mechanism for
browsing any network systems and resources that you may be able to
connect to in a way that is independent of the actual type of network
vendor. Traditionally, if a system needed to be simultaneously connected
to different types of network, the way in which each could be connected
and viewed would be vendor-specific. Windows NT Workstation 4 is
capable of displaying a common view of your entire network even though
it may actually comprise resources from Windows NT, Novell NetWare,
Banyan Vines, or others!
The Inbox icon is used if Microsoft Exchange is active on your system.
Windows NT Workstation 4 has built-in electronic mail services based on

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Microsoft Mail (MS Mail) and Microsoft Exchange. If there is already an

MS Mail post office on the same network that the system is connected to,
the Windows NT Workstation 4 mail client can connect directly to it. The
Inbox lets you access your messages.
The Recycle Bin icon represents the holding place for deleted items. As
long as files are in the Recycle Bin they can easily be recovered if they
have been accidentally deleted. Windows NT Workstation 4 will preserve
files until the system runs out of free disk space. When this happens
Windows NT Workstation 4 will prune the contents of the Recycle Bin on
a first-in first-out basis.
Caution
Files that are overwritten due to an application using a duplicate filename

will not be saved to the Recycle Bin.
Double-clicking the Bin displays its contents. The empty window confirms
that there are no items in the Recycle Bin.
The Start Menu

A single click of the left mouse button on the Start button opens the start
menu and presents the seven major option categories for starting work
on the system.
A single click of the right mouse button opens a small but powerful
control menu containing the options Open, Explore and Find.
Their functionality is described below.
Starting a Program
The Start menu contains the various categories where your applications
and work are stored. You can move further into the various
subcategories by positioning the mouse over the category that you are
interested in to automatically open the next subcategory. You do not
have to click the mouse!
The Programs command displays the Programs menu. This menu lists
all of the applications installed and available to you. Some options are
marked by an arrow. This arrow indicates that a submenu follows. Drag
the mouse cursor over the Accessories command to see its submenu.
The Accessories submenu lists the set of Windows NT built-in programs.
TIP: If you drag an object either from the Desktop or Windows Explorer
and drop it directly onto the Start button, a link to that object will
automatically appear in the Start menu.
There are many ways to start a program. The following method is the
simplest one:
1. Click on the Start button.
2. Click on Programs.
3. Click on the group that contains the program you want to start (for
instance, LCS).
4. Click on the program you want to start (for instance, Leica LCS).
Another way to start a program is to open a document that you created in
that program. The program automatically opens when the document
opens. Double-click on a document file in My Computer or Windows
Explorer to open it. Or click on the Start button and select a recently used
document from the Documents menu.
You can also start a program by double-clicking on its shortcut icon on
the desktop. Shortcut icons are links to other files. When you use a
shortcut, Windows simply follows the link back to the original file.
Whenever you use a document or program frequently, you might
consider creating a shortcut for it on the desktop. To do so, just use the
right mouse button to drag an object out of Windows Explorer or My

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Computer. After releasing the mouse button, a menu appears. Select the
option Create Shortcut(s) Here. Some programs automatically create a
shortcut during their installation procedure.
Caution
Windows NT Workstation 4 does not actively track a link between an

original and a shortcut. For instance, if you create a shortcut of a
program, and subsequently move (rather than copy) the original to a
different folder, then the shortcut may no longer function.
The Startup folder is special in one respect: any programs located in this
folder will start automatically when Windows NT Workstation 4 is started.
The Documents menu shows the names of the 15 files you created most
recently. You can open any of these files and its related application at the
same time by clicking the file's name in this menu.
Caution
Document files that are opened within an application (typically by

selecting the File/Open command within the application) will not be
displayed here. Only documents opened directly from the Desktop will be
displayed here.
The Settings menu offers three commands for changing your system's
settings. You can directly access the Control Panel and Printing folders.
Also accessible is the Task Properties window.
Being able to access the core system configuration utilities in this way is
particularly useful when an application is already in the foreground and
you want to make a quick change.
The Find command features an easy way to locate all system resources.
Within the Find category you can perform searches for three distinct
types of search as described below:
The Taskbar
The taskbar–positioned at the bottom of the screen–provides a constant
view of which applications are running on the system and an easy way of
switching between them. As you add to the number of concurrently
running applications, the taskbar automatically re-sizes its iconized view
of the applications to ensure that they can always be seen. To switch
from one running program to another, simply click on the second
program as displayed in the taskbar.
The taskbar also provides constant additional information such as the
system time and volume control if a sound card is installed. All of these
functions can be adjusted by the user.
Setting the Time and Date

The current Date, Time and Time Zone information can be set from the
Date/Time icon within the Control Panel. This setting is important since
Windows NT stamps the date and time on all of your files as you create
and modify them. The two options can be selected by clicking on the
appropriate tab.
To change the Date and Time
Click on the appropriate date or use the controls to change the month or
year. The time can also be changed by first selecting the digital display
and then using the up and down arrows.
To change the Time Zone
Select the appropriate Time Zone from the drop down list at the top of the

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screen. Notice that the option to automatically adjust the clock for
daylight savings time is selected. On some systems you can also drag
the highlighted area on the world map and drop it on the correct location.
Changing the date and time information within Windows NT Workstation
4 will update the battery backup CMOS clock in your system.
Note
Depending on the shell configuration, systems connected to a network

may get a time and date update from a network server every time they
log on. If the server's time is incorrect, your workstation's time will be
wrong, too. Please inform your network administrator.
Getting Help
Windows NT includes a powerful help system. In addition to Help menus
in every window, there is a standalone Help feature available from the
Start menu. To access it, click your mouse on the Start button, and click
on Help.
There are three tabs in this box: Contents, Index, and Find. The Contents
tab appears on top. To move to a tab, click on it.
Contents
The Contents tab displays individual Help topics. The topics are
organized into categories and are represented by small book icons.
Double-click on any book to open it. Sub-books and documents appear.
Double-click on sub-books and documents to open them.
Index
The Index tab displays an index of all available topics. Type the word you
want to look up. The Index list scrolls to that part of the alphabetical
listing. When you see the topic on the list that you want to read, double-
click on it.
Find
Rather than searching for information by category, the Find tab offers a
full text search. Enter the word(s) or phrase to be searched for under
Help in the text entry box. The text entry box is linked to a list of words in
your Help files, and any words or phrases that match will be displayed.
You can specify more than one word by separating words with a space. If
you wish to change a search option, select Options. The first time you
click on this tab, Windows tells you it needs to create a list. Click Next
and then Finish to allow this. The main Find tab appears next. Type the
word(s) you want to find in the top text box. Then click a word in the
middle box to narrow the search. Finally, review the list of help topics at
the bottom, and double-click the one you want to read.
When you're done reading about a document, click Help Topics to return
to the main Help screen, or click Back to return to the previous Help
topic. Click the window's Close button to exit Help.
Shut Down Windows NT

Always use the Shut Down command before you turn off your PC. The
Shut Down option allows the user to close the Windows NT Workstation
operating system and ensure all running processes can halt cleanly and
are given the chance to flush any data out to the disk that may be in
cache memory. Several options are available when shutting the system
down.

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Caution
Powering down your computer without prior shutting it down may result in
severe data loss.
Setting Up Users Under WinNT

1. Logging on as administrator
Log on as administrator by using the ID "Administrator" and the password
"Admin."
2. Opening the User Manager
Select Start → Programs → Administrative Tools → User Manager.
3. Defining a new user
Enter at least the following information in the opened dialog window:
1. User name
2. Password (must be re-entered in the following line for confirmation
purposes)
Select the following two check boxes:
a.) "User must change password at next logon" (this allows the new user
to define his or her own password at logon)
b.) "Password never expires" (this allows a defined password to be valid
either until it is changed in the User Manager or the user is deleted)
Select the "Profiles" option in the bottom section of the dialog. In the
"Local path" field enter the following path for storing user-specific files:
d:\users\username ("username" is an open parameter which must be
replaced by the currently defined user name).
Note
Factory installed hard disks feature 2 partitions (C:\ and D:\).The user
directory should be set up on partition D:\.

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The Leica Confocal Software:

Overview
Starting the Software

Requirements for Starting the Software
The LCS software is copy-protected to prevent it from being used on two
computers at the same time. This protection system allows all additional
application packages to be used. The protection system consists of a
dongle that must be inserted into the parallel port of the workstation. The
functionality of the parallel port (e.g., for printing, etc.) is not affected. To
use the software on a second computer, the dongle must be attached to
its parallel port.
Note
If you remove the dongle from the workstation of the confocal system, the
software cannot be started, thereby preventing operation of the confocal
system.
The LCS software can be started in two operating modes: hardware
mode and simulation mode. In hardware mode, all hardware components
are accessed and initialized by the software. For this reason, you should
switch on the hardware first and then wait about 20 seconds before
starting the software.
In simulation mode, the software runs independently of the hardware.
This mode is intended for secondary installations on another computer,
for example for training or offline analysis of existing data.
Starting the Software
Select Start|Programs|Leica Confocal Software. The initial screen of
the Leica Confocal Software appears. This window allows you to select
from three profiles.
Company
This option starts the Leica Confocal Software using the default factory
settings. This means that the configuration and the position of the
Toolbars is preset. These settings cannot be changed.
Personal
This option lets you use a user-defined configuration profile. The user
name is dependent upon the account used to log on to the operating
system. If the custom profile does not yet exist at the first initialization,
the default factory settings are applied to the personal profile.
Last Exit
This option loads the configuration profile that was last active.
For advanced users:
If you have more than one configuration profile, you can load them at
startup by clicking the button with the three black dots, which is located at
the right lower edge of the profile options. You may also use this option
to reset your custom configuration profile to the standard factory profile.
Clicking the Start button starts the Leica Confocal Software using the
corresponding configuration profile.
Note
After a delay, the software starts automatically and loads the currently
selected configuration profile.

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The Experiment Software Concept

The Leica Confocal Software allows you to join image data or edited
images into groups. Each group is called an "experiment" and is stored in
a special data format (*.lei). This makes it possible to store both the
original experimental image data and the image viewing data together.
Additional information can be found in the chapter "Organizing Data by
Grouping Experiments"
The Basic Structure of the User Interface

The appearance of the Graphical User Interface–henceforth referred to
as GUI–depends strongly on the configuration profile being used. A
number of elements of the GUI, however, are standard.
The GUI provides the following standard elements:
The menu:
The menu contains the submenus File, View, Macro, Tools, Window and
Help. The submenus contain commands and information for general
display, settings and user customization. They do not provide any of the
functions for directly controlling the scan functions. These functions are
located in the TCS menu (View → Menu → TCS Menu). The menu line
cannot be customized.
The Viewer window (TCS_Viewer):
The Viewer window displays the image data, experimental conditions and
user data. The image window can be configured (see the chapter on
"Modifying the User Interface and Defining User-Specific Settings"). The
image window shows not only the confocal image data records, but also
the experiment data and system settings. You can open a new
experiment image window by selecting File → New.
The TCS menu (TCS_Menu):
The TCS menu contains buttons for the individual device functions. It is
subdivided into individual work steps. The number of work steps
available depends on the installed software. The standard set of work
steps consists of data recording (Acquire), image viewing (View), surface
reconstruction (3 D), measurement functions (Quantify), image
processing and analysis functions (Process), and documentation
functions (Annotate). If the TCS menu is not displayed using the current
configuration profile, you can activate it by selecting View → Menu →
TCS Menu.
The toolbars:
This area allows you to insert and customize individual (function) buttons.
The advantage of the container is that it can be switched on or off with its
entire contents of buttons. To do so, select View → Menu → Container.
The document viewer window (Experiment Overview):
The document viewer window displays the recorded experiments and
their contents in a directory tree. Open this viewer window by selecting
View → Experiment Overview.
The status bar:
The status bar is found at the bottom edge of the Leica Confocal
Software user interface. It displays:
• the progress while loading image data (progress bar)
• the version number of the software
• the name of the system configuration (system type)
For details about these functions, refer to the chapter on "Software
Reference".

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Opening Data Records

Readable File Formats
The following file formats can be opened and viewed in the Leica
Confocal Software:
Experiment (*.lei):
The format of this type of file is Leica-specific and binary. This format is
provided for the data of entire experiments.
Tiff files (*.tif):
These are Leica image files in single and multi TIFF format. Both image
files in the previously used TCS formats and external files in RGB TIF
format can be read.
Annotation (*.ano):
The format of this type of file is also Leica-specific and binary. It is used
for saving annotation sheets. The elements on the annotation sheet,
such as images, texts and graphic images, are each available as
individual objects.
When these files are loaded, both the image data and the experiment
settings are loaded.
Automatically Applying the Study Parameters of an Experiment
With the Leica Confocal Software, hardware settings that have been
saved for previous experiments or single images can be applied to new
experiments. This allows you to carry out different experiments using
constant settings. To carry over settings from a previous experiment,
open the viewer window for the data record that contains the settings you
want to apply. There, click the "Apply" button, which, in the factory default
configuration profile, is located in the toolbar.
Note
If you do not find the "Apply" button in one of the open windows, you can
load it into any of the windows by selecting Tools → Customize. In the
dialog window that appears, select the "Commands" tab under the File
category. With the left mouse button, click the "Apply" button and drag it
while holding the mouse button pressed to the desired window. Release
the left mouse button to insert the button at the current location.
Saving Images
As described above under "Readable File Formats", individual images
and experiments can be saved using the same data formats.
Select File → Save to save your images and experiments. The first time
an experiment is saved, the "Save as" dialog opens automatically,
allowing you to specify a file name. In addition to defining a suitable file
name, you can also select a file format here. Experiments can only be
saved in the Leica-specific *.lei format. When you are saving
experiments, you may be able to save existing individual images in *.tif or
*.raw format.
Note
If you are saving an experiment or image that has already been saved
before, the old data will be overwritten.If you do not want to do this,
select File → Save as to save the new or changed data to a new file with
a new name.

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Data Organization by Grouping Experiments

The concept of the Leica Confocal Software allows you to group
individual images, image series and the results of edited images into
single groups, or experiments. The Experiment Overview window
provides a general overview of the open experiment. To open the
Experiment Overview window, select View → Experiment Overview.
Create a new experiment by selecting File → New or File → New
(Template). Files that were previously saved are handled as separate
experiments when opened.
Compiling Experiments
After you have defined a new experiment by selecting File → New or
File → New (Template), you can start filling it with data.
Note
Images that have been recorded in continuous scan mode are

overwritten automatically when the next scan is started. If you want to
keep a single recording as a permanent part of an experiment, you
should use the single scan function.
An experiment contains data acquired with the "single scan" or "series
scan" function. If you carry out image processing functions on a data
record, you can save the results as additional components of the
experiment. To do this, double-click the desired image or series in the
experiment overview window. Carry out the desired image processing
functions (such as maximum projection or topological image or others).
Mark the region within the viewer window that you want to keep as part of
the experiment. Click the right mouse button (to open the context menu)
and select Send to → Experiment. The Selection (raw) option stores a
copy of the raw data of the selected object as a new, separate
component of the experiment. The Selection (snapshot) option stores
an RGB image (no 3D data, just a pure snapshot) of the selected object
as a new, separate component of the experiment.
Menu Functions
The following TCS SP2 functions can be carried out from the menu bar:
File Menu
New:
This option opens a new experiment in a new window. The display
properties of the new window are determined by the active defaults (see
the chapter on "Default Settings and Templates").
New (Template):
This option opens a new experiment in a new window. The way in which
the viewer window is displayed can be selected from a set of templates.
Open:
This option opens a previously saved experiment or a single data block
(image data or annotation). The display properties of the new window are
determined by the active defaults (see the chapter on "Default Settings
and Templates").
Open (Template):
This option opens a previously saved experiment, a single data block or
annotation files. The way in which the viewer window is displayed can be
selected from a set of templates.
Close:

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This option closes the current experiment. It does not save the
experiment automatically before closing it.
Note
You are not asked whether you want to save the experiment before it is
closed.
Close all:
This option closes all open experiments. It does not save the experiment
automatically before closing it.
Note
You are not asked whether you want to save the experiment before it is
closed.
Save:
Saves the current experiment.
Recent files:
Opens one of the recently opened files.
Print:
Opens a dialog for printing the content of the currently active window.
Exit:
Terminates the program.
The View Menu

Menus:
Use this option to switch TCS menu and the toolbar on and off.
Status bar:
This option switches the display of the status bar on and off at the bottom
edge of the user interface of the LCS software.
Experiment Overview:
This option opens a window displaying the individual experiments and
their components (data records, graphs, etc.) in the form of a tree.
Double-click an experiment component within the Experiment Overview
window to bring the corresponding viewer window to the foreground.
Viewer Options:
Use this item to configure various options for displaying data records in
the viewer window, such as the display angle and calculation methods for
displaying image data, etc.
The Macro Menu

Macros:
Depending on the software installed, either use this option to edit macros
directly or just list and run them.
Note
You can edit and modify macros only if the complete, integrated VBA
development environment (IDE) is installed.
Record a New Macro:
This starts the automatic macro recorder. For more information, refer to
the documentation provided with the optional macro development
environment.
Pause Recording:
This pauses the macro recorder.
Stop Recording:
This stops the macro recorder.
Visual Basic Editor (optional):
This option launches the software for developing macros and programs

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based on Visual Basic for Applications (VBA). Here you can edit and
modify macros and even develop entire programs.
The Tools Menu

Legend Info:
This option opens the dialog window for entering user-specific data. This
data (e.g., name of institute, name of specimen, etc.) is saved with each
data record and can be displayed next to the acquired images in the
legend.
Objective:
This option opens the dialog window for defining applied objectives. The
objectives you use can be made known to the software by copying them
using "drag and drop" from the list provided in this dialog window.
Note
It is important to apply the correct data for the objectives, as some of the
calculated sizes (such as the scan field dimensions) depend on them.
Microscope:
Use this option to select the Leica microscope you are using. This setting
influences the way in which the microscope is controlled (for example,
the motorized objective nosepiece).
Settings:
Use this option to select the hardware settings that you want to be
applied when opening a previously saved data record.
License:
This option allows you to install subsequently purchased licenses. A
subsequent license usually is installed if a software package offered as
an option is purchased.
The Window Menu

New Window:
This option opens a new viewer window within the same experiment. For
example, you can use this function to view the same data record using
different views (such as topographical and overlay views).
The Help Menu

Contents:
Opens the table of contents of the online help.
Search:
Starts the search function for the online help.
Index:
Opens the index of the online help.

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Keyboard Shortcuts
Special key combinations have been defined to speed up access to
frequently used software functions.
Keyboard Function
Shortcuts
F1 Opens the online help.
ALT + F8 Opens the Macros dialog window for running, editing and
deleting macros.
ALT + F11 Starts the VBA development environment (optional).
(optional)
CTRL + L Opens the Legend Info dialog window for entering user-
specific settings that can be applied when documenting,
saving and viewing the documentation of image recordings.
CTRL + J Opens the Objective dialog window for defining and selecting
applied microscope objective lenses.
CTRL + N Opens a new experiment.
CTRL + O Starts the Open dialog window for opening an existing file.
CTRL + P Opens the Printer Selection dialog window.
CTRL + S Saves the active experiment.

Introduction to the Leica Confocal Software Help
Three different help levels are available for the Leica TCS SP2:
Quick Help
When you let the mouse pointer hover over a Leica Confocal Software
button, a brief explanation of the function of this button is displayed. This
so-called Help Banner automatically disappears when the mouse pointer
is moved.
Context-sensitive Help
If you want to use the context-sensitive help function, click on the Help
button:
The entire user interface is then frozen and a question mark appears
beside the mouse pointer. Then, instead of triggering the button function,
clicking a button opens an explanation of the button's function. If the Help
button is not present on the user interface:
! Select the Customize option from the Tools menu. Here you will find all
buttons arranged by categories.
! The Help button can be found in the File category.
! Click on it using the left mouse button and drag it to the desired window.
Contents of the Online Help

Select the Contents option from the Help menu to view the online help
table of contents, which allows you to select any function in order to view
information on it.

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Keyword Search (Index)

Select the Index option from the Help menu to view an index of key
words. Select a key word. View the corresponding content pages by
double-clicking the key word or selecting it and then clicking the Display
button.
Full-text Boolean Search

Select the Search option from the Help menu to launch the full-text
search engine. Enter your search word in the input field. Click the triangle
to the right of the input field to view the available logical operators. Select
the desired operator. Enter the second search word you would like to
associate with the first search word behind the operator:
Examples Result
Pinhole AND This phrase finds help topics containing both the word
Sections "pinhole" and the word "sections".
Pinhole OR This phrase finds help topics containing either the word
Sections "pinhole" or the word "sections" or both.
Pinhole This phrase finds help topics containing the word "pinhole" and
NEAR the word "sections" if they are located within a specific search
Sections radius. This method also looks for words that are similar in
spelling to the words specified in the phrase.
Pinhole NOT This phrase finds help topics containing the word "pinhole" and
Sections not containing the word "sections".
Favorites
If you select Favorites in the dialog window of the online help tab, you
can include the current help topic in a list, making them easily available
for future use.
Opening the Context-Sensitive Help
Function
Click Help to open the context-sensitive online help function, which
provides you with short explanations for the various buttons and
functions of the Leica Confocal Software.
! Click the Help button.

! A question mark appears next to the mouse pointer. This temporarily
disables the functions of all buttons.
! Use the mouse pointer to click the button that you want an explanation of.
! Online help opens directly to the description for the corresponding button.
Online help also provides you with an index of key words and a search
function so that you can search for specific topics and buttons.
Furthermore, you can print the individual descriptions.
You can also open the online help by selecting Contents, Search or
Index option under the Help menu.

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Documentation Conventions
Button
The buttons provided on the user interface of the Leica Confocal
Software. Buttons are marked with icons and/or have an (often
abbreviated) English label. They either trigger actions directly or open
dialog windows.
Menu
The menus are divided into the categories of File, View, Macro, Tools,
Window and Help and are displayed in the menu bar located at the upper
edge of the user interface.
Option
Options refer to the selectable items that are hierarchically listed below
the menus. Options either trigger actions directly or open dialog windows.
Dialog Window
Both buttons and options open dialog windows. Dialog windows are used
to set various parameters and select functions.
Register
Registers are found in dialog windows. Registers thematically combine
the parameters and functions that can be configured in dialog windows.
Some registers are divided into fields.
Viewer window
The Leica Confocal Software contains two types of viewer window. The
Viewer window is called up by pressing the New button and displays the
recorded images. The Experiment Overview viewer window displays the
recorded images in a directory tree. This viewer window is called up from
the View menu and appears as a separate window at the left side of the
user interface.
Legend
The Leica Confocal Software provides two legends, which display the
parameters and settings of an image recording. The Experiment legend
can be placed at the right edge of the Viewer window. The Hardware
legend is called up from the View menu and appears as a separate
window at the left side of the user interface.
Context Menu
Context menus appear when you click the right mouse button while
holding the mouse pointer over certain areas of the user interface.
Context menus contain various, context-sensitive commands.
! see ...
This symbol is a reference to another topic in the online help.

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Data Recording
Setting the Beam Path
Function
Open the Beam Path Setting dialog window using the Beam button, and
set up the beam path and the detectors with the corresponding color
look-up tables used to record the image.
Selecting the excitation wavelength

Above the spectrum in the dialog window you will find boxes to set the
excitation wavelength using an AOTF:
! Click the check box next to the laser name to activate the laser.
! Set the output for the laser line by moving the slide on the corresponding
scale to the desired value, or
! Double click the percentage value for the laser output. This opens a
second dialog window where you can enter an exact value.
! The active laser line is shown as a line in the spectrum.
Loading and Saving Parameter Settings

Also above the spectrum is a list box for loading and saving parameter
settings. The manufacturer has predefined parameter settings for the
most important fluorescent dyes. They are labeled with the letter L (for
Leica) and can be loaded only, not modified.
You can also save the settings made for a specific image recording as
user-defined parameter configuration so that you can load them any time
with a single click:
! Click the Save button.

! This opens a dialog window for entering a name for the parameter
configuration.
! The parameter configuration is stored in the list box under User and is
labeled with the letter U (for User).
Furthermore, you can individually select the parameters that you want
saved when using the Save command:
! Select the Settings option from the Tools menu.

! In the Settings dialog window, click the Instrument Parameters tab.
! Select individual parameters by clicking the corresponding checkboxes.
The Select all and Deselect all buttons allow you to select all or no
parameters quickly.
Click one of the user-defined or preset parameter configurations and then

press the right mouse button to open a context menu, which contains the
following commands:

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Command Function
Set as default The standard setting for the parameters is loaded when
setting you start the software.
Remove default This removes the default setting property from the
setting selected configuration.
Load Loads the parameter configuration.
Rename (only for Use this to change the name of the parameter
U) configuration.
Delete (only for U) Deletes the parameter configuration.
Selecting the excitation beam splitter

The symbol (green, tilted line) for the excitation beam splitter is located to
the left of the spectrum. Click on the symbol and select the desired beam
splitter:
! Neutral filters, such as the RT 30/70 filter, are used in reflection

applications to lead the excitation light to the sample and the reflected light
to the detection pinhole.
! Dichroic filters, such as the reflection short pass filter RSP 510, are used in
fluorescence applications to split and detect fluorescent light of a particular
wavelength range from the excitation light.
! Double dichroic filters are used for image recording, where the preparation
is marked with two fluorescent dyes and two excitation wavelengths. The
DD 488/568 double dichroic, for instance, is used to split excitation light
with a 488 nm and 568 nm wavelength from the detection light. Depending
on the system configuration, you can also select DD 488/ 543 and DD 458/
514 type double dichroites.
! Triple dichroic filters are used for image recording, if the preparation is
marked with three fluorescent dyes and three excitation wavelengths. The
TD 488/568/633 triple dichroic, for instance, is used to split excitation light
with a 488nm, 568nm, and 633nm wavelength from the detection light.
Depending on the system configuration, you can also select TD 488/ 568/
647 and TD 488/ 543/ 633 type triple dichroites.
Setting detectors and color look-up tables (LUT)

Below the spectrum in the dialog window the boxes for the four detectors,
PMT 1 to 4, as well as for the PMT Trans transmitted light detector are
shown. Adjust the settings as follows:
! Activate each of the desired detectors by clicking on its corresponding

Active check box below the color look-up table icon. A cast shadow now
links the activated detector to the corresponding slide bar on the scale of
the spectrum.
! Specify the wavelength range for the detection by dragging the two ends of
the slider to the desired positions on the scale, or
! Double-click the slide bar to open an additional dialog window in which you
can enter a precise value for the start point and the end point of the
wavelength range.
! For fluorescence applications, list fields with common fluorescence dyes
have been set up for each detector. Select the desired fluorescence dye to
display its emission curve in the spectrum.
! Select a color look-up table by clicking on the corresponding icon.
! see Selecting Color Look-Up Tables

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Selecting an Objective
Function
Click the Objective button to open a list of objectives and make an
appropriate selection. This list contains only the objectives that were
previously assigned to one of the maximum of seven threaded ports on
the objective nosepiece. You can assign objectives as follows:
! Select the Objectives option from the Tools menu. A dialog window opens
containing an extensive list of objectives and the symbolic representation
of the slots on the objective nosepiece.
! Find in the list the objective that you are using and select it with the mouse.
Click and hold the left mouse button and drag the objective onto the
symbol representing the slot in which the objective is installed.
! The assignment is saved in the software and the objective appears in the
selection list, which you can open by pressing the Objective button.
! Repeat this procedure for every objective that you have installed in the
objective revolver.
You can use the Add, Remove and Edit buttons in this dialog window to
add new objectives, remove objectives or edit existing objective labels.
Note
The microscope models DM RXA, DM RXE, DM IRBE, and DM IRBE2

use a software program to control the objective nosepiece. The program
automatically rotates the selected objective into the beam path whenever
you select an objective by using the button or Objective dialog window.
All other microscope models require not only software adjustment of the
objective, but also manual rotation of the objective into the beam path.
Additional information
When selecting the correct objective for a specific application, the
objective's correction class (achromatic, apochromatic, fluorite objectives
and plane objectives) and especially the magnification factor and the
numerical aperture are of great significance. The numerical aperture
determines the resolution capacity of an objective and is deduced from
the flare angle of the light cone recorded by the objective and the
refraction index of the medium between objective and specimen: NA =
n*sin a
Objectives with greater magnification generally have larger numerical

apertures but smaller entrance pupils and therefore can record light only
from a relatively small scan field. Objectives with larger apertures permit
higher resolutions but allow less free working distance. The following
table illustrates this relationship:

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Objective Resolution Resolution Resolution Resolution Scan

(xy) Air (z) Water (z) Oil (z) Field Size
(xy)
HC PL 1301 19410 25879 29559 3000
FLUOTAR 5x
0.15
HC PL 651 4768 6407 7335 1500
FLUOTAR 10x
0.30
N PLAN 20x 488 2630 3566 4093 750
0.40
N PLAN 50x 260 649 948 1108 300
0.75
PL APO 100x 139 319 209 236 150
1.40
Values in nm at Values in
wavelength λ=488 mm
nm
Typical Applications
Material scientists commonly use dry objectives to study surface
structures. Immersion Objectives are the best choice for producing
images of layered structures in which material layers with varying
refraction indexes come into contact with each other. When working with
biological specimens, the choice between an oil immersion objective and
water immersion objective depends on the specimen itself and its
embedding medium. You will achieve the best resolution when the
refraction indexes of the embedding medium and/or the specimen are
matched to the refraction index of the objective medium.
Setting the Detectors
Function
Use the Signal button to open a dialog window for adjusting the detectors
so that the entire range of intensities of a color look-up table is assigned
and displayed in the image. For this purpose, a Gain value and an Offset
value can be set for each detector. The gain value modifies the
amplification of the detected signal, thus changing the brightness and
contrast of the image. The offset value defines a threshold value. Only
those signals that lie above the threshold value are detected and
displayed in the image.
There are two methods for setting the gain and offset values:
! Use the mouse to move the slide on the scale. The corresponding value is
shown below the corresponding scale.
! Double-click the numerical value that is displayed below the scale. This
opens a second dialog window where you can enter an exact value.
Best suited to optimization of gain value and offset values are the color
look-up tables Glow Over, Glow Under and Glow Over and Under.
! see Selecting Color Look-Up Tables

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You can also set the gain value and offset value of the detectors using
the corresponding radio buttons of the control panel.
! see Controlling Functions Using the Control Panel
Note
A detector is not enabled unless its corresponding Active checkbox is

checked in the Signal or Beam Path Setting dialog windows.
The Electronic Zoom
Function
In confocal microscopy, the magnification of an image is determined both
by the objective and the electronic zoom. The objective generates a first
image whose magnification is based on the objective's magnification
factor. The electronic zoom provides additional magnification. A zoom
factor of 1 scans the maximum scan field size with a specific number of
dots. If the zoom factor is set to 2, the same number of dots is used to
scan a scan field with half the page length of the maximum scan field (1/4
the original scan field). This achieves stronger magnification and thus
better image resolution too, because a smaller scan field is scanned with
the same frequency, which results in a higher density of data.
In the dialog window that was opened by pressing the Zoom button, you
can select one of the preset zoom factors. If you click on the Others
button, you can set a different zoom factor in two ways:
! Use the mouse to move the slide of the scale. The corresponding value is
shown in the middle of the dialog window.
! Double-click the boldface numerical value located in the middle of the
dialog window. This opens a second dialog window where you can enter
an exact value.
You can also set the zoom factor using the corresponding rotary knob on
the control panel.
! see Controlling Functions via Control Panel
While zoom factors from 1 to 32 can be set, the useful magnification of
the electronic zoom cannot be increased infinitely. The limit is achieved
using the smallest optically resolvable distance, which is determined by
the resolution capacity of the objective. According to the Nyquist
theorem, this smallest optically resolvable distance can only be mapped
without information loss if it is scanned with about 2 to 3 raster points. If
the scan frequency is exceeded at a relatively high zoom factor and the
specified scan format, further magnification is no longer useful because
no more optical details can be resolved (zero-order magnification).
!see Selecting a Scan Format

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! see Selecting an Objective

Note
With bleach-sensitive specimens it is advisable to restrict the use of the

electronic zoom to short time intervals. Since you are recording a
segment of the specimen with a relatively higher scan frequency in the
case of high zoom factors, the specimen is exposed to a stronger impact
of light. This can lead to photomechanical destruction of the specimen
(optical bleaching).
Optimal Zoom Factors

The following table shows which zoom factors (marked red) - depending
on the scan field size and scan format specified by the objective that can
be used to scan the specimen without loss of data:
Scan Zoom Scan Scan Resolution/

Objective Format Factor Field Frequency Scan
s Size (nm) Frequency
(mm)
Magnificatio Numerical Resolution
n Aperture at λ=488
nm
20 0,6 325 1024 x 1 750 732 0,4
1024 2 375 366 0,8
4 187 183 1,8
6 125 122 2,7
512 x 512 1 750 1465 0,2
2 375 732 0,4
4 187 365 0,9
8 94 183 1,8
10 75 146 2,2
200 x 200 1 750 3750 0,1
2 375 1875 0,2
4 187 935 0,3
8 94 470 0,7
16 47 235 1,4
32 23 115 2,8
40 1,25 156 1024 x 1 375 366 0,4
1024 2 187 183 0,8
4 94 92 1,7
6 62 60 2,6
512 x 512 1 375 732 0,2
2 187 365 0,4
4 94 183 0,8
8 47 92 1,7
10 37 72 2,2
200 x 200 1 375 1875 0,1
2 187 935 0,2
4 94 470 0,3
8 47 235 0,7
16 23 115 1,3
32 12 60 2,6
100 1,4 139 1024 x 1 150 146 0,9
1024 2 75 73 1,9
3 50 49 2,8
512 x 512 1 150 293 0,5
2 75 146 0,9
4 37 72 1,9
6 25 49 2,8
200 x 200 1 150 750 0,2
2 75 375 0,4
4 37 185 0,7
8 18 90 1,5
12 12 60 2,3

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Note
The Leica Confocal Software provides three different zoom functions: the
electronic zoom, the 3D zoom and the graphic zoom.
! see Zooming the 3D View

! see Viewer Options Dialog Window, Display Icon
Enlarged Recording of a Frame
Function
The Zoom In function enables an enlarged recording of a freely
selectable square frame of a specimen.
! Click the Zoom In button. At the same time, the Rectangle button is
automatically activated.
! see Defining the Region of Interest (ROI) as a Rectangle
! Specify the frame to be enlarged by drawing a rectangular evaluation area
(region of interest or ROI) into the image using the Rectangle button.
! see Selecting and Moving the Region of Interest (ROI)
! Use the Select button to move the frame on the image.
! To start the image recording, press the Continuous Scan, Series Scan or
Single Scan button.
Note
The software automatically adapts the freely selected rectangular frame

to the actual square form that can be implemented by the scanner. The
dashed line shows the actual frame.
The enlarging recording of a frame will only provide additional detailed
information as long as the distance of the grid points remains larger than
half the optical resolution. On the other hand, the resolution depends on
the objective used, the adjusted scanning format, and the light
wavelength used (Empty magnification).
All applications that deal with the optical resolution of very small
structures and that can do without an overview image. While recording
such specimens, care should be taken that an objective with a
corresponding high optical resolution is used for image recording.

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Setting the Detection Pinhole
Function
Clicking the Pinhole button opens a dialog window that you can use to
set the diameter of the detection pinhole. In the upper right list field in the
dialog window, select the units for displaying the diameter. You can
select from mm, Airy units and digital values. There are two ways of
specifying a value for the diameter:
! Use the mouse to move the slide on the scale that is displayed on the left
in the dialog window. The corresponding value is shown in the middle of
the dialog window.
! Double-click the boldface numerical value located in the middle of the
dialog window. This opens a second dialog window where you can enter
an exact value.
The diameter of the detection pinhole must be set to the currently used
objective at all times. If you click the Airy 1 button, the detection pinhole
is set automatically to the optimum value of 1 Airy unit depending on the
objective in use.
In addition to the numerical aperture of the objective and the wavelength
of the light, the detection pinhole also determines the thickness of the
optical sections.
The diameter of the pinhole has the optimum if it matches the diameter of
the Airy disc. The Airy disc refers to the inner, light circle of the diffraction
pattern of a point light source. The diameter of the Airy disc, in turn, is
also dependent on several optical parameters and can be described for
the Leica Confocal System as follows:
To calculate the diameter of the Airy disc, you need the excitation
wavelength l (in the case of several wavelengths, a mean value should
be used); the numerical aperture NA and the magnifying factor M of the
objective. The factor of 3.6 refers to the magnification of other optical
components belonging to the Leica Confocal microscope.
If the pinhole is set to the Airy disc, light from outside the focal plane is
suppressed and the signal-to-noise ratio is high. These conditions allow
the recording of optical sections of minimum thickness. The wider the
diameter of the pinhole, the more light reaches the detector. The image
becomes brighter but blurring from structures outside the focal plane will
also appear in the image, making it increasingly unfocused.
Increasing the diameter of the pinhole above 1 Airy unit is recommended

only for detecting very weak signals.
Generally when recording material scientific specimens, enough light is

reflected into the detectors so that the detection pinhole can be closed
completely (i.e. set to the minimum diameter).

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Selecting a Scan Format
Function
Clicking the Format button opens a dialog window containing a selection
list of scan formats. Selecting the scan format selects the image raster
that will be used for recording the images. An image raster is the amount
of points scanning the specimen in the three directions in space. Besides
the numerical aperture of the objective and the excitation wavelength, the
scan format, together with the electronic zoom, determines in large part
the spatial resolution of the recorded data.
When selecting the scan format, observe the influences between the
image raster and the resolution of the generated image. According to the
Nyquist theorem (or sampling theorem), a structure can only be scanned
without information loss if the smallest optically resolvable distance is
scanned with about 2 to 3 raster points. This optically resolvable
distance, called lateral resolution, depends on the numerical aperture of
the objective and the wavelength of the applied excitation light:
The following example should illustrate this relationship: You have

selected, for example, the PL APO 100x objective unit with a numerical
aperture of NA = 1.4 and a wavelength of l = 488 nm. Based on these
factors, the smallest optically resolvable distance is determined as
follows:
Based on this rough formula, the distance of the raster points required
avoiding information loss while recording is:
If however you select a scan format of 1024x1024, a PL APO 100x

objective, which has a maximum
Scan field size of 150 mm, would result in the following raster point
distance:
To achieve the required 47 nm raster distance, you can either raise the
scan format (to 2048x2048 for instance) or you can decrease the scan
field size using the Electronic Zoom (Zoom=2).
The current raster distance is displayed in the Hardware legend. This

value is referred to as Voxel Size and recalculated as soon change the
scan format, the Electronic Zoom or the objective.
! see The Electronic Zoom


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Note
When the sampling rate of 2 to 3 points is exceeded, it is referred to as

oversampling. In the case of oversampling, the structure is fully mapped
but no additional information is gained. The disadvantage of
oversampling is the long scanning time and the danger of destroying
bleach-sensitive specimens. When the sampling rate is less than 2 to 3
points, it is referred to as undersampling. In this case, there is the danger
that not all necessary information is sampled. The image might illustrate
structures that are not actually present in the specimen. This effect is
called aliasing.
Selecting a Scan Mode
Function
Clicking the Mode button opens a dialog window containing a list of
available scan modes. The scan mode determines which optical levels of
the specimen are to be scanned. Fundamentally, horizontal xy-sections
or vertical xz-sections can be recorded. To generate a three-dimensional
image of the specimen, the optical sections are continued in the
corresponding third direction in space, thus recording a stack of
individual images. In addition to this, it is possible to add the factor of
time or wavelength while recording the images:
Mode Function
xyz An image stack is recorded from the xy-sections in the z direction.
xzy An image stack is recorded from the xz-sections in the y direction.
xt A line is recorded several times in succession.
xyt A xy-section is recorded several times in succession.
xzt An xz-section is recorded several times in succession.
xyzt An image stack is recorded several times in succession from xy-
sections in z-direction.
xyλ An xy section is recorded at different wavelengths.
xzλ An xz section is recorded at different wavelengths.
All scan modes (with the exception of xt) are composed of at least three
dimensions. The device will ignore the third as well as other dimensions if
you record images only from one optical plane (xy or xz) using the
Continuous Scan function of the Single Scan function.

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Note
The xzy and xzt scan modes are only displayed in the list if you
previously selected the selection point Galvo using the Z-Scan button.
Likewise, the scan modes for a xyλ and xzλ wavelength will only be
displayed if only one detector is activated in the Beam Path Setting
dialog window.
! see Setting the Beam Path
Selecting a Scan Speed
Function
Clicking the Speed button opens a dialog window that you can use to
select from four scan speeds.
Speed
200 Image lines per second
The data recording speed is further increased in combination with the bi-
directional scan.
! see Unidirectional or Bi-directional Scan
The higher the set scan speed, the shorter the dwell time of the laser
point. Furthermore, the scan format, i.e. the number of sampling points in
a row, needs to be taken into account. The higher the scan format at a
constant speed is, the shorter the dwell time of the laser point over a
sampling point.
The longer the light point of the laser beam dwells over the individual
sampling points of the specimen, the more light is detected by the
detector. So using a lower scan speed results in a better signal-to-noise
ratio. The disadvantage of a lower scan speed is that the relatively long
impact of the light on the specimen can bleach the specimen
photochemically, making it unusable. This is especially important with
fluorescence applications
Note
If the speed levels 800 or 1000 are set, technical limitations prevent the
maximum scan field from being scanned. The system automatically
switches to zoom factor 2 or zoom factor 4.

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Setting the Z/Y-Position
Function
Use the z/y-Position button to define the horizontal level (z position) or
vertical level (y position) of the specimen in which the images are to be
recorded. To record an image series using the Series Scan function,
define the start and end points of the image series using the z/y-position
button and the Begin and End buttons.
! see the Series Scan Overview Dialog
Clicking the z/y-Position button opens a dialog window that provides you
with two ways of specifying a position value:
! Use the mouse to move the slide on the scale that is displayed on the left
in the dialog window. The corresponding position value is shown in the
middle of the dialog window.
! Double-click the boldface position value located in the middle of the dialog
window. This opens a second dialog window where you can enter an exact
value.
The z/y-position can also be set using the corresponding knob on the
console.
! see Controlling Functions via Control Console
Configuring a Time Series
Function
Click the Time button to open the Time Configuration dialog window for
setting up a time series recording. The settings that can be changed in
this dialog window depend on the selected scan mode. You can record a
line (xt), a horizontal section (xyt), a vertical section (xzt), or a stack of
horizontal sections (xyzt), interrupted by a particular time interval, many
times in a row.
Note
The Time button is not enabled unless you have selected a scan mode
with time dimension using the Mode button.
! see Selecting a Scan Mode

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Use the following parameters in the dialog window to configure a time

series using the xt scan mode.
∆T The recording time for a line (cannot be modified by the user)

Lines Number of line recordings
Lines per Number of lines per stored page
page
Pages Number of stored pages (calculated automatically)
Complete Total recording time, i.e. the product of ∆T and number of
Time recordings
Using the Calculate button, you can calculate how many stored pages
are needed for a particular number of lines per page.

series using the xyt or xzt scan mode.
∆T Recording time for a xy-section or xz-section plus pause

interval
Frames Number of recordings of the xy-section or xz-section
Time recordings

series using the xyzt scan mode:
∆T Recording time for a stack of xy-sections plus pause interval

Stacks Number of recordings of the image stack
Time recordings
Each parameter can be calculated in relation to the other parameters.

When entering the values, note how the dialog window operates. The
entry field you click on will be activated:
! Click on the parameter to be calculated. The corresponding entry field is

grayed out.
! Now enter the values for the other parameter.
! Click on Apply to calculate the parameter. By clicking on Reset, the last
saved values are displayed.
When you have finished configuring these settings, press the Series
Scan button.
Starting a Single Scan
Function
Use the Single Scan button to record a single image from a single optical
level in the specimen.
Before recording an image using the single scan function, configure all

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required scan parameters using the Continuous Scan function to ensure

optimal image quality.
! see Starting a Continuous Scan
The single scan function is designed for recording bleach-sensitive
specimens. Use this function in the case of bleach-sensitive specimens
not only for image recording, but also as an alternative to the continuous
scan function for setting the scan parameters. The single scan function
can also be used to check the image section by zooming in on the scan
field.
Starting a Continuous Scan
Function
Use the Continuous Scan button to permanently record images from a
single optical level in the specimen. This does not generate image series,
since the image being generated always replaces the previously
generated image.
Press the button a second time to stop the continuous scan.
The unit applies the last used scan parameters automatically. You can
modify some of the parameters while the image is being recorded.
Others have to be configured before you start the recording:
Before Recording While Recording

Selecting an Objective Adjusting the electronic zoom
Selecting a Scan Mode Setting the Detector
Selecting a unidirectional or bi-directional scan Adjusting phase
Before Recording While Recording

Selecting an Objective Setting the Beam Path
Selecting a Scan Format Setting the Detection Pinhole
Selecting a Scan Mode Adjusting the electronic zoom
Selecting a Scan Speed Setting the Detectors
Selecting a unidirectional or bi-directional scan Adjusting phase
Setting the z/y Position
The buttons of the functions that cannot be changed during continuous

scanning are disabled and appear gray.
The continuous scan function is best used to optimize the image quality
for the first scan of a specimen. While the specimen is being scanned
continuously, you can modify the scan parameters listed above and
check the results directly in the image.

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Note
With bleach-sensitive specimens it is advisable to restrict the use of the

Continuous Scan function to short time intervals. Subjecting the
specimen to the continuous light of the laser can destroy the specimen
photochemically (optical bleaching), thus making it unusable.
Series Scan Overview Dialog Window
Function
This dialog window lets you define the begin and end points of an image
series and trace the recording of the individual sections. The three-
dimensional scan area is represented graphically as a cube. Within this
cube, a yellow square represents the current z position or y position, a
green one represents the begin point and a red one the end point. The
corresponding position values are displayed to the right of the cube. Set
the begin and end point as follows.
! Use the mouse pointer to drag the yellow square to the level in which the
image series is to begin.
Or click the z/y-Position button to open a dialog window where you can
enter the values of the z position or y position.
! Click in the white box for the begin point. The corresponding position value
appears and is saved.
! Use the mouse pointer to drag the yellow square to the level in which the
image series is to end.
Or click the z/y-position button to open a dialog window where you can
enter the values of the z-position or y-position.
! Click in the white box for the end point. The corresponding position value
appears and is saved.
! The entire height of the image stack between the begin and end points is
calculated and displayed (total).
Now click the Series Scan button. The dialog window stays open and you
can follow the process of the image series being recorded.
Note
The start point and end point can also be defined using the control panel
and the separate keys Begin and End.
! see Defining the Start Point for a Spatial Series

! see Defining the End Point for a Spatial Series
! see Setting the z/y Position
The following additional parameters, which have to be set before
recording an image series, are specified in the Series Scan Overview
dialog window:

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! Scan mode (top line to the right of the graphic image)

! Scan format (red digits at the upper edge of the cube)
! Number of optical sections (division between begin and end points)
The red number on the vertical edge of the cube represents maximum
travel of the z-actuator that cannot be changed.

! see Selecting a Scan Format
! see Defining the Number of Spatial Sections
Defining the Begin Point for a Spatial Series
Function
Use the Begin button to define the begin point of a spatial image series.
First, set the exact z-position and/or y-position using the z/y-position
button or on the control panel using the corresponding radio button. Then
click Begin. This saves the position value for the start point. The end
point is set in the same manner.

Note
You can also conveniently set the begin point and end point of a spatial
series in the Series Scan Overview dialog window. Open this dialog
window by pressing the Series button (the small button, not the button
that starts the Series Scan function).
! see The Series Scan Overview Dialog
Defining the Begin Point for a Wavelength Series
Function
For a wavelength series, this function records a stack of individual
images, each of which are detected at a specific wavelength, from a
single, optical level. Use the Lambda Scan Begin button to define the
wavelength at which recording should begin:

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Note
The Lambda Scan Begin button is only active if you used the Mode
button to select a scanning mode with the wavelength dimension.
! Press Beam to open the Beam Path Setting dialog window.

! Activate a detector by clicking its corresponding check box. A cast shadow
now links the activated detector to the corresponding slide bar on the scale
of the spectrum.
! Double-click this slide bar to open the Range Properties dialog window,
where you can specify the end point of the series.
! Drag the slide bar on the scale of the spectrum to the desired start
position. The left edge of the slide bar represents the wavelength at which
the recording is to begin.
! Click Lambda Scan Begin to save the value.
The end point of a wavelength series is set in the same manner. In

addition, you also have to set the number of wavelength steps.
! see Defining the End Point for a Wavelength Series

! see Setting the Number of Wavelength Steps
A wavelength series can be applied to determine the maximum emission
of a fluorochrome. This is useful because the Stokes shift of the emission
curve of a fluorochrome is dependent on each specimen that is applied.
This then allows you to set the detection range precisely for a specific
application.
Defining the End Point for a Spatial Series
Function
Use the End button to define the end point of a spatial image series.
First, set the exact z position or y position with the z/y Position button or
with the corresponding rotary knob on the control panel. Then click End.
This saves the position value for the end point. The begin point is set in
the same manner.

Note
You can also define the begin and end points for a spatial series in the
Series Scan Overview dialog window. Open this dialog window by
pressing the Series button (the small button, not the button that starts the
Series Scan function).

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! see The Series Scan Overview Dialog
Defining the End Point for a Wavelength Series
Function
single, optical level. Use the Lambda Scan End button to define the
wavelength at which recording should end:
Note
The Lambda Scan End button is only active if you used the Mode button
to select a scanning mode with the wavelength dimension.
! Press Beam to open the Beam Path Setting dialog window.

! Activate a detector by clicking its corresponding check box. A cast shadow
now links the activated detector to the corresponding slide bar on the scale
of the spectrum.
! Double-click this slide bar to open the Range Properties dialog window,
where you can specify the end point of the series.
! Drag the slide bar on the scale of the spectrum to the desired end position.
The right edge of the slide bar represents the wavelength at which the
recording is to end.
! Click Lambda Scan End to save the value.
The begin point of a wavelength series is set in the same manner. In

addition, you also have to set the number of wavelength steps.
! see Defining the Begin Point for a Wavelength Series

application.

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Defining the Number of Spatial Sections
Function
Clicking the Sections button opens a dialog window that you can use to
select the number of horizontal xy-sections and vertical xz-sections for
recording an image series. If you want to specify a number of sections
that is not listed, click the Others option. This opens the Z/Y
Configuration dialog window, which contains the following data:
Parameter Description
Image Dim. z/y The height of the entire image stack between the begin and
(mm) end points of the image series
# Sections The number of configured sections
Step Size (mm) The step size, i.e. the distance between two sections
In this dialog window, you can enter any value for the number of sections
and the section width. The height of the image stack cannot be changed,
as this parameter is determined by the start and end point of the image
series. As the section width always has to be a multiple of the minimum
section width of the z-setting drive, certain combinations of values are
only possible if either the height of the image stack or the number of
sections is adapted. Depending on which of the two Calculate buttons
you click, one of the two parameters is not changed.
If you want to calculate the number of sections with the priority of leaving
the height of the image stack unchanged where possible:
! Enter the desired step size in the Step Size field.

! Then click the Calculate button next to the Step Size field.
If you want to calculate the number of sections with the priority of leaving
the number of sections unchanged where possible:
! Enter the desired step size in the Step Size field.

! Then click the Calculate button next to the # Sections field.
If you want to calculate the step width with the priority of leaving the
height of the image stack unchanged where possible:
! Enter the number of desired sections (whole numbers only) in the #

Sections field.
! Then click the Calculate button next to the # Sections field.
If you want to calculate the step width with the priority of leaving the
number of sections unchanged where possible:
! Enter the number of desired sections (whole numbers only) in the #

Sections field.
! Then click the Calculate button next to the Step Size field.
If you click the Reset button, the last shown values are displayed.

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! see Starting a Series Scan

! see Series Scan Overview dialog window
Setting the Number of Wavelength Steps
Function
single, optical level. The images are recorded within a wavelength range,
which is limited by the begin and end points. Use the Lambda Steps
button to define the number of recordings that are to take place within
this range.
Note
The Lambda Steps button is only active if you used the Mode button to
select a scanning mode with the wavelength dimension.

application.
Starting a Series Scan
Function
Use the Series Scan button to create an image series. This creates a
multidimensional image data block of the specimen. The available
dimensions for recording an image series are the three directions in
space (x, y, z) as well as the dimensions of time (t) and wavelength (λ).
This allows you to record a three-dimensional, spatial image stack
consisting of xy or xz-sections with the additional factor of time or
wavelength.

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Note
Before recording an image series, configure all required scan parameters

using the Continuous Scan function to ensure optimal image quality.
! see Starting a Continuous Scan
Before recording a spatial image series, configure the following settings:
! Select the scan mode using the Mode button.

! Select the scan format using the Size button.
!see Selecting a Scan Format
! Set the desired z-position or y-position using the z/y-position button or the
corresponding knob on the console.
! see Controlling Functions Using the Console
! Set and store the begin point for an image series using the Begin button or
in the Series Scan Overview dialog window.
! see Defining the Begin Point for a Spatial Series
! Set and store the end point for an image series using the End button or in
the Series Scan Overview dialog window.
! see Defining the End Point for a Spatial Series
! Select the number of spatial sections using the Sections button.
! see Defining the Number of Spatial Sections
Scan button.
You can track the recording process of the image stack in the Series
Scan Overview dialog window.
Before recording a time series, configure the following settings:
! Select a scan mode with time dimension using the Mode button.
! Set the number of recordings, the pause interval between the recordings,
and the complete editing time.
! see Configuring a Time Series
Before recording a wavelength series, configure the following settings:
! Select a scan mode with wavelength dimension using the Mode button.
! Define the wavelength at which you want the wavelength series to begin.
! Define the wavelength at which you want the wavelength series to end.

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! Select the desired number of recordings between the begin and end points
of the wavelength series.
Scan button.
Selecting a unidirectional or bi-directional scan
Function
If you click the Unidirectional/ Bi-directional Scan button, bi-directional
scan mode is enabled. If this button is not clicked, unidirectional scan
mode is automatically set.
In unidirectional scan mode, each line is scanned from left to right. No

data is recorded while the laser beam is being guided to the starting point
of the next line. In bi-directional scan mode, the first line is scanned from
left to right and the second from right to left. In other words, the return
run, or flyback, of the laser beam is implemented for recording data too,
thus increasing the scan speed when using bi-directional scan mode.
If bi-directional scan mode is active, you can double the scan speed
using the Speed button:
Unidirectional Bi-directional
200 Currently not available Image lines per second
400 800 Image lines per second
! see Selecting a Scan Speed
In order to accurately align the pixels of the forward sweep and flyback,
the phase between the forward sweep and the flyback can be adjusted.
Use the Phase button or the appropriate knob on the control panel.
!see Setting the Phase

! see Controlling Functions using the Control Panel
Note
If the speed levels 800 or 1000 are set, technical limitations prevent the
maximum scan field from being scanned. The system automatically
switches to zoom factor 2 or zoom factor 4.
!see Electronic Zoom

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Adjusting phase
Function
During bi-directional image recording, there may be a phase
displacement between the scanning beams moving forward and
backwards. Click on the Phase button to open a dialog window that
allows you to correct the displacement:
! Use the mouse pointer to move the slider on the scale until the pixel
displacement disappears from the image.
It is also possible to adjust the phase with the respective knob on the
control panel.
! see Controlling Functions using the Control Panel
Before delivery of the instrument, the factory performs a phase
adjustment for each zoom value and enters the corresponding values.
Due to the temperature dependency of the mechanics and electronics,
there may be a slight deviation from these default values during
operation. Use this function to readjust these values.
Turning the Scan Field
Function
Use the Scan Field Rotation button to rotate the scan field. This neither
rotates the specimen nor the direction of the scanner, but rather the
intermediate microscopic image.
The angle of rotation is infinitely variable between -10 and +100 degrees.
The original position (0 degrees) is based on a predefined reference
position in the scanning head. When the unit is switched on, it
automatically relocates to this reference position.
This function is used to align textures on surfaces or long stretched-out
biological structures relative to the viewer.

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Recording an Image of a Line Using the Averaging

Method
Function
The Line Average function launches an averaging method for the image
recording. In this case, each single line is scanned several times. For
every sampling point, the arithmetical average is calculated from the
repeatedly measured intensity values in one line and represented in the
result image. The next line of the specimen will not be scanned until after
the set number of averaging steps has completed. The method used
here determines a consecutive average. This means that every line
recorded after the first line is averaged with the previously displayed line
and is displayed in the result image (dynamic average).
Clicking the Line Average button opens a dialog window that you can use
to set how often a line is to be scanned. You can select from 1 to 8 scan
repeats.
Using a parallel function, it is possible to record complete images using

the averaging method.
! see Recording an Image Using the Averaging Method
This function is used predominantly for recording live specimens.
Note
Recording an image using the averaging method is not recommended for

bleach-sensitive specimens. Repeated recordings and the resulting
length of subjection to light can lead to the destruction of the specimen.
Applying the Parameter Setting of an Experiment
Function
Click the Apply button to use the hardware settings of a previous
experiment for recording a new experiment. This lets you configure the
settings for new image recordings with a single click with the scan
parameters that have been optimally configured for a previous
application.

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! Activate the image data block that is configured with the settings you want
to apply to the new experiment.
! Click the Apply button.
Recording an Image Using Burst Mode
Function
If the workstation must process large amounts of data during the
recording of images, it may lead to a delay of the scanning process. This
is because the recorded image is first displayed on the monitor before
the scanner can scan additional images. The Burst function allows for
decoupling the scan process of the laser and the refreshing of image
data on the screen. For this purpose, the transmission of image data
from the program memory to the monitor is delayed, but the scanning
process is not. By clicking on the Burst button, you can select operating
modes with different delay times:
Mode Description
No Burst Image data are recorded and simultaneously and continuously
displayed on the monitor.
Frame Burst Image data are not displayed on the monitor until a single
image has been recorded.
Complete Image data are not displayed on the monitor until an image
Burst series has been recorded.
Automatic The software automatically sets the optimum-operating mode
for a specific application.
Note
The following parameters can increase the amount of data to be

processed to the extent that it will delay the display on the monitor:
Scanning format, scanning speed, bi-directional scan, number of active
detection channels, calculation of an overlay image, and size of the
Viewer window.
Recording an image using the Averaging Method
Function
The Average function launches an averaging method for the image
recording. In this case, every individual image, i.e. every xy-section or xz-
section, is scanned several times. For every sampling point, the
arithmetical average is calculated from the repeatedly measured intensity

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values and represented in the result image. The method used here
determines a consecutive average. This means every image recorded
after the first image is calculated with the results of the previously
displayed image and is displayed in the result image (dynamic average).
Clicking the Average button opens a dialog window that you can use to
set how often a section is to be scanned. You can select from 1 to 64
scan repeats.
Recording an image using the averaging method is primarily useful for
suppressing noise. In fluorescence microscopy, weak fluorescent
specimens result in little light reaching the detector. The resulting low
photon count leads to noisy images. In such cases, you can improve the
signal-to-noise ratio by carrying out multiple recordings and determining
averages of the image.
Note
Recording an image using the averaging method is not recommended for

bleach-sensitive specimens. Repeated recordings and the resulting
length of subjection to light can lead to the destruction of the specimen.
Image Recording With a Digital Resolution of 8 Bits

or 12 Bits
Function
This function reads in the analog intensity values measured by the
detector by means of an A/D converter, either as 8 bit signal or 12 bit
signal.
While digitizing the analog intensity signal with a digital resolution of 8 bit,
256 different intensity values can be displayed. Taking the natural
statistical variations of the typical intensity value range of fluorescence
specimen into account, a digitization with 8 bit is completely sufficient in
most cases for an image recording without loss of information. For
specimens with a higher intensity dynamics (e.g., for specimen with very
intensity-weak and intensity-strong areas), an image recording with 12 bit
digitization is recommended. In a 12-bit digitization, 4096 different
intensity values can be resolved.
For image recording with very high intensity dynamics (mostly material
specimens with high reflectivity of the surface and occurrences of very
dark areas at the same time).

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Setting the UV Lens Wheel
Function
For each objective, this function reduces the focus offset between visible
and UV excitation light to a value below the optical resolving capacity. In
order to achieve this, a suitable correction lens is brought into the beam
path for each objective suitable for use with UV excitation light. To use
this corrective feature, proceed as follows:
! Click the Objective button (standard procedure in Acquire step).

! Select a UV-capable objective from the list (see indication on list).
In this case, the corrective lens for the selected objective is automatically
moved into the beam path.
Note
The possible occurrence of a focus offset between visible light and UV

light has physical reasons and is not a malfunction of the device.
Other Requirements
In order to achieve an automatic correction of the physically based color
length fault between visible and UV light, it is not sufficient to manually
rotate the objective into the beam path.
Data Viewing
The Viewer Window
In the default setting, the Viewer window consists of three important

areas. The image window (4), in which the recorded images are
displayed, is located in the middle. Button pads (1) and (2) can be
arranged to the left and below the image window and the Experiment
legend (3) to the right of it. It is possible to select other default settings of
the Viewer window and to save user-defined configurations as a
template.

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! see Saving Viewer Window Viewer as a Template
Button pads (1) and (2)

By default, the button pad to the left of the image window contains the
buttons for image viewing functions and the button pad below the image
window contains the buttons used for scrolling through the individual
images of an image series. Both button pads can be moved within the
Viewer window or removed from it as separate windows. To do so, click
on the double-lined edge of the button pad and drag it while keeping the
mouse button depressed to the desired position.
Image window (4)

If you place the mouse pointer at any position within the image window
and then press the right mouse button, a context menu containing the
following commands opens:
Command Function
Send to Experiment Selection This copies the raw data of a
(raw) selected area in the image window
as a new image in the current
experiment. The new image can be
processed.
Selection This creates a new image in the
(snapshot) current experiment from the
snapshot of a selected area in the
image window. The new image can
no longer be processed.
All This creates a new image in the
(snapshot) current experiment from the
snapshot of the entire, current
image window. The new image can
no longer be processed.
Printer Selection This prints the area selected in the
image window.
All This prints the entire, current image
window.
Left This shows or hides the button pad to the left of the image
buttons window.
Bottom This shows or hides the button pad located below the image
buttons window.
LUT This shows or hides the color look-up tables of the current
image.
Legend This shows or hides the Experiment legend.
Full screen This enlarges the Viewer window to full screen.
Viewer This opens the Viewer Options dialog window.
Options
The color look-up tables (5) are displayed as color bars to the right of
the image window. If you hold the mouse pointer over a color bar, grab
points appear at the top and bottom of the color bar. You can use these
grab points to limit the current color look-up table to a specific intensity
range and to load a second color look-up table. This allows you to
increase the contrast of the image graphically.

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! Drag the top grab point down or the bottom grab point up.
! Double-click the area above or below the corresponding grab point.
! The Select LUT's dialog window opens so that you can select a second
color look-up table.
! The upper and lower intensity ranges are represented in the colors of the
second color look-up table.
! see Selecting Color Look-Up Tables (LUT)
The Experiment (3) Legend

Various image parameters of an image recording are registered in the
Experiment legend. To select the parameters that you want displayed,
click any area of the legend. This opens a list of all available entries.
Then select the desired entry. If you hold the mouse pointer over any
area within the Experiment legend and press the right mouse button, a
context menu containing the following commands opens:
Command Function
Experiment Edit This opens the Edit Legend dialog window, where
you can specify a (Title) for the legend, specify the
(Number of legend entries) or (Clear all entries).
Activate This displays the Experiment legend in the Viewer
window.
Remove This deletes the current Experiment legend.
Add This creates a new Experiment legend in the Viewer window.
Experiment
tab
The Hardware Legend

The hardware settings for image recording are registered in the
Hardware legend. To open the legend, select the Hardware Legend
option from the View menu. To select the entries that you want displayed
in the legend, click the Edit button. This opens the Edit Legend Entries
dialog window:
! The Available Entries list box shows all available entries. Select the entries
that you want displayed in the legend. Then click Add to carry the selected
entries over into the Show entries list box.
! The Show entries list box contains the entries that are to be displayed in
the legend. Use the Remove button to remove entries from being displayed
in the legend.
! Use the Move up and Move down buttons to shift one or more than one
entry up or down in the list.
! You can use the Edit grid color and Edit background color buttons to
modify the color of the frame and background of the legend.
The Hardware legend is aligned automatically along the right edge of the
user interface. You can change however the size and position of the
legend as desired:
! To change the width of the legend, use the mouse pointer to drag the edge
to the position you desire.
! To change the position of the legend, double-click on the double-lined
edge of the legend or click the 5 icon once. This releases the legend from
the user interface, turning it into a window. Now you can drag the legend to
any position by pressing and holding the left mouse button.

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Note
If the legend has been enlarged to cover the entire width of the user
interface, you first have to shrink the height of it in order to drag it back to
the edge of the user interface.
Option New Window

You can open a second Viewer window containing the same image as
the currently open Viewer window by clicking the New Window option in
the Window menu. This command does not create a new experiment, but
rather opens a second view of the current image data. This feature is
useful for viewing different representations of the same image data at the
same time. Sequential numbers are added behind the file extension to
distinguish between the resulting copies of the current Viewer window.
Viewer Options
Viewer Options Dialog Window, 3D Icon
Function
To open the Viewer Options window, select the Experiment Overview
option in the View menu.
The Experiment Overview window appears on the left side of the user
interface. The top part of the viewer window displays the recorded
images in a directory tree. The bottom part displays the Viewer Options
window. This dialog window is used to perform basic settings for different
software functions. The left side shows the icons corresponding to the
functions and the right side shows the related tabs. When you open the
dialog, it contains the icons of the functions that you are currently using.
Click Show all to view all icons.
The tabs of the 3D icon can be used to perform optional settings for the
following functions:
! see Rotating the 3D View

! see Moving the 3D View
The Navigation tab displays the numerical values of the actions that are
carried out using the Rotate, Move and Zoom buttons (and the mouse
pointer).
Use the Rotation field to tilt a 3D view in all three spatial directions by
modifying the angular degrees of the three axes. The 3D view is rotated
around a fixed point, which is located in the center of the image. To best
understand the rotation function, modify the angle of one axis at a time,
while leaving the other two axes set to 0.

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Rotation Function
X from 0° to The 3D view is rotated 45° around the fixed point in the
45° direction of the negative z-axis.
Y from 0° to The 3D view is rotated 45° around the fixed point in the
45° direction of the negative y-axis.
Z from 0° to The 3D view is rotated 45° around the fixed point in the
45° direction of the negative y-axis.
Use the Translation field to shift a 3D view to the right or left, up or

down and enlarge or shrink it by modifying the coordinate values. (Users
of all regional versions should be sure to use the dot as the decimal
placeholder.)
Translation Function
X Positive values shift the 3D view to the right, negative to the left.
Y Positive values enlarge the 3D view, negative shrink it.
Z Positive values shift the 3D view up, negative down.
Use the Predefined field to change the viewing perspective of the image
to Top view or Side view by clicking these buttons respectively.
In the Display tab click on one of the listed commands to show or hide
the corresponding graphic element of the 3D view in the Viewer window:
Graphic Elements Function

Show LUT Show the selected color look-up table on the z-axis.
Show scale Show the scale.
Show bounding box Show the bounding box, which limits the measurement
volume.
Show axes Show the coordinate axes.
Show data during Show the image data while the 3D view is being rotated,
3D motion shifted, enlarged or shrunken.
Viewer Options Dialog Window, Display Icon
Function
The tabs of the Display icon can be used to perform optional settings for
the following functions:
! see Starting and Ending a Film

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On the Settings tab, use the graphic zoom to enlarge or reduce the
image displayed in the Viewer window:
n-1 Displaying n pixel as 1 pixel in the resulting image reduces the

image.
Automatic The image is displayed in the original image format.
1-n Displaying 1 pixel as n pixel in the resulting image enlarges the
image.
Note
graphic zoom, the 3D zoom and the electronic zoom.

! see Electronic Zoom
Click on one of the selection points on the same tab to hide or display the
respective graphic element in the Viewer window:
Coordinates The z position of the current image is displayed.

Scale A measurement bar with length indicators is displayed.
Grid A grid is placed over the current image.
The length of the measurement bar and the grid width are calculated
dependent on the objective, the electronic zoom and the beam
divergence.
You can use the Movie tab to set the speed for running the film
sequence of an image series. You can choose speeds from 6 images per
minute to 25 images per second.
! Use the mouse to drag the slide on the scale to the desired value.
! Select the Ping-Pong mode if the film sequence is to run from the first to
the last image and then in reverse order back to the first image. If this
mode is not selected, the film sequence always starts with the first image
again.
Viewer Options Dialog Window, Charts Icon
Function

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The tab of the Charts icon can be used to perform optional settings for
! see Measuring a Profile Along a Line Segment
! see Measuring a Profile Within a Region of Interest (ROI)
! see Measuring Surfaces and Volumes
! see Measuring Roughness Along a Line Segment
The options in Charts tab can only be selected if one of these buttons
was used earlier to activate a quantification function.
Activating the two-point measurement
! Clink on 2 Point in the Measurement field to activate the two-point

measurement.
Each of the buttons listed above opens a window that displays a

measuring curve and statistical values.
If the two-point measurement is selected, the window shows a
measurement slider (2) with two measurement points (3) below the
measurement curve (1). The position of the two measurement points in
the profile curve is symbolized by small black squares (4). This allows for
very precise settings of the range to be evaluated within the
measurement. In addition, the statistics shown below the measurement
curve is expanded by the following measurement values: position of
measurement points, distance of measurement points, measurement
value at the measurement points, difference of the measured variable
between the measurement points.
Changing the position and length of the measurement slider
! To change the position, move the mouse over the slider, click and hold the
left mouse button and drag it to the desired position.
! To change the length of the slider, grasp it at one end with the mouse and
enlarge or reduce it while pressing the left mouse button.
Changing the scaling of the measurement curve

By default, the scaling of measurement curves is automatically adapted
by the software to the intensity value range that is present in an image
recording. However, the scaling can also be changed manually:

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! Deselect the Auto check box to deactivate automatic scaling.

! You can now redefine the upper and lower limit value of the value range
separately for each measurement curve (each detection channel).
! Check the results of the changed scaling directly in the window.
Viewer Options Dialog Window, Online Measure

Icon
Function
In the tab of the Online Measure icon, you can make optional settings for
The slider on this function can be used to set the number of optical
sections for which the measured values of an online measurement are
displayed graphically. The developing trend in an experiment can be
seen from the online measurement graph.
Note
The trend data only exists in temporary form and it cannot be saved. The
temporarily saved data can however be exported via the context-
sensitive menu (right mouse button) or copied into an annotation sheet.
! see Exporting Quantification Data

! see Copying Quantification Data to the Annotation Sheet
Quantitative evaluation of data records

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Viewer Options Dialog Window, Projections Icon
Function
In the tabs of the Projections icon, you can make optional settings for the
following functions:
! see Maximum Projection of an Image Stack With Invariable Projection

Angle
! see Average Projection of an Image Stack With Invariable Projection
Angle
! see Transparent Projection of an Image Stack With Invariable Projection
Angle
! see Creating an SFP Projection of an Image Stack
The Projections tab contains various options for generating a projection

image. Select whether the intensity maximums, the arithmetical average
or a weighted average of the intensities are to be displayed in the
projection image. Vary the weight factor for the transparent projection.
Set a threshold value or scaling factor for the displayed intensities.
Type Function
Maximum From each of the examined columns of sampling points, the
projection sampling point with the highest intensity is displayed in the
two-dimensional projection image as the representative of all
values within the column.
Average From each of the examined columns of sampling points, the
projection arithmetical average of the intensities measured in the
column is displayed in the projection image.
Transparent From each of the examined columns of sampling points, a
projection weighted average of the intensities measured in the column
is displayed in the projection image.
Transparent Function (enabled only for transparent projection)

factor
Drag the slide on The higher transparent factor a (0 < a < 1) setting, the
the scale. stronger the intensity values flow from the lower levels in
the image stack into the projection image.
Threshold Function (enabled only for average and transparent

projection)
Drag the slide on Intensities that are below the specified threshold value
the scale. are ignored in the calculation of the projection image.

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Scaling Function (enabled only for average and transparent

projection)
Rescale to The measured intensity values are scaled to the maximum
maximum possible intensity values.
The SFP tab contains various options for generating an SFP projection
image.
! see Principles and Types of Projections
Field Function
Light Here, enter a coordinate for x and y for the projection angle of
Direction the SFP projection. This angle symbolizes the angle of
incidence of the laser beam onto the specimen. Click on the
Apply button to create the projection with the new angle.
Absorption You use the slider to vary the absorption coefficient a. The
higher this is set, the smaller the values calculated in the
projection image. If the coefficient is set as low, many of the
pixels in the projection image reach the maximum value, making
it increasingly difficult to distinguish between the structures.
Threshold Use the slider to define a threshold value. The intensity values
below this value are not taken into account for the creation of
the projection image.
Rescale to The intensity values of the image are scaled to the maximum
maximum possible intensity values. This ensures that the calculated gray
values of the projection image remain within the value range of,
for example, 0 to 255 at 8 bits. This allows for brightening dark
images.
Viewer Options Dialog Window, Overlay Icon
Structure of the Experiment Overview window

The tab of the Overlay icon can be used to perform optional settings for
! see Viewing an Overlay Image
On the Overlay tab you can select among three types of creating an
overlay image from raw data and the corresponding color look-up tables:

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Color Merging Function

True Color (add The color values of the pixels in the original images
RGBs) are averaged and displayed in the overlay image.
True Color (add The color values of the pixels in the original images
RGBs) with dynamic are averaged, scaled to the maximum possible color
adjustment values and displayed in the overlay image.
Fast (bit wise "OR" The color values of the pixels in the original images
RGB) are mixed using a fast bit-by-bit calculation process
and displayed in the overlay image.
! see Boolean Operations
Coloring Function
Only Red/ The color look-up tables Red, Green and Blue are always used
Green/ Blue to create the overlay image, independent of the current color
look-up tables of the original images.
Viewer Options Dialog Window, Scan Progress Icon
Function
In the tab of the Scan Progress icon, you can make optional settings for
The currently set scan mode is shown in the Scan Progress tab. The
status of an image recording can also be checked on the basis of the
progress display.
Viewer Options Dialog Window, Surface View Icon
Function

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In the tab of the Surface View icon, you can make optional settings for
! see Creating the 3D View
In the Visualization tab, you can select whether the 3D view is to be

generated as Surface, Wireframe or Isolines and specify the perspective
in which the 3D view is displayed:
Render Function
Mode
Surface The blank spaces between the pixels are filled in with surface.
Wireframe All pixels are linked with lines, while the blank spaces remain
free.
Isolines Pixels that correspond to values of the same intensity are
enclosed in a curve.
Projection Type Function

Perspective The 3D view is displayed in central perspective.
Parallel The 3D view is displayed in parallel perspective.
The Stretch height (factor) field offers the ability to vary the scaling
factor in the z-direction, thus stretching or shrinking the height of the 3D
view.
Use the Downsample rate field to reduce the density of data of the 3D
view to increase the speed of image processing. At a pixel density of 1:1,
all measured intensity values are displayed in the image. At a pixel
density of 1:2, only every second measured intensity value is used in the
surface image.
Use the Isoline interval field to define a distance for separating the
individual isolines in mm. This allows you to limit the number of isolines in
the 3D view.
Use the Isoline detail level field to enter a limiting value, specifying that
only isolines of a particular length are to be displayed in the 3D view.
This limits the displayed isolines to those, which correspond to an
intensity value that has a specific frequency of occurrence.

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Viewer Options Dialog Window, Surface Measure

Icon
Function
In the tab of the Surface Measure icon, you can make optional settings
for the following functions:
In the Leveling field, there are options for an adaptation function to bring
measurement curves of a roughness profile that show a trend into a
horizontal position. For this purpose, a linear interpolation function
(polynom) is fitted to the measurement curve.
! see Image Tool Dialog Window, Basic Button

! see Adaptation Function for Correction of Trends
None No adaptation function is applied to the measurement curve.

Automatic The measurement curve is brought into a horizontal position
linear using a linear adaptation function.
Interactive The position of the measurement curve is corrected using the
level linear adaptation function in such a way that the line segment
between the two measurement points is on the horizontal plane.
Freeze The newly positioned measurement curve is locked so that the
item of the measurement points can be changed again without
interactively modifying the measurement curve.
The Multipoint Measurement field provides the possibility of saving the

two measurement points as well as the difference in height between the
measurement points.
! Click on the Remember button to save the values. The Clear button
deletes them again.

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Viewer Options Dialog Window, Surface Calculation

Icon
Function
In the tab of the Surface Calculation icon, you can make optional settings
for the following functions:
! see Creating a Topographical Image
The Topography tab contains various options for generating a

topographical image. Select whether the intensity maximums or centers
of mass of the intensities are to be interpreted as height in the
topographical image. Specify the type of visualization for height in the
topographical image and set a threshold value for the displayed
intensities.
Surface Function
Reconstruction
Search maximum From each of the examined columns of sampling points,
intensity the sampling point with the highest intensity is displayed
in the two-dimensional topographical image as the
representative of all values within the column.
Calculate center of From each of the examined columns of sampling points,
mass of intensities the center of mass of the surface, which is restricted by
the curve of the measured intensity values, is displayed
in the two-dimensional topographical image.
Topography Function
Processing
Invert height The column of sampling points is examined in reversed
direction. This reverses the height information. A negative
of the topographical image is displayed.
Level The horizontal position of a recording is corrected using a
linear interpolation function (polynom).
Threshold Function
Drag the slider on the Intensities that are below the specified threshold
scale or enter a numerical value are ignored in the calculation of the
value. topographical image.

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Image Tool
Image Tool Dialog Window
Schematic Structure of the Dialog Window

The numerous functions of the Image Tool dialog window are arranged
on a number of levels. The gray buttons 3D, Basic, Amplitude, Materials
and Multicolor on the left side of the dialog window represent the first
level of the hierarchy. Each of these buttons has a group of icons that
appear below the selected button. The icons are the next level on which
functions are grouped. Each icon opens different tabs and fields that are
displayed on the right side in the dialog window and arrange the
functions on the lowest level.
The following elements can be found in each tab:
! In the Source Image drop-down list at the top of the dialog window, select
the image data record to be processed.
! If you click on the Use Selected Image check box, the image data record
currently displayed in the Viewer window is used for the calculation.
! If you click on the Show button at the bottom of the dialog window, the
selected calculation is activated and the result image is displayed in the
preview window in the dialog window.
! If you click on the Apply button at the bottom of the dialog window, the
Viewer window and created as a new file in the current experiment.
! Click on the button with the arrow symbols to show the preview window on
the right edge of the dialog window or to hide it. The projections triggered
using Show are displayed temporarily in this preview window.
! The Close button closes the dialog window.
! see Creating an Experiment
Overview of the Function Groups Created in the Image Tool Dialog

Window
Button Icon Tab Description of Function

3D Projections & Projections Creating projection images
Animations Rotation along a variable projection
Stereo axis
View Running Projection Images as
3D Rotation Animation
Creating red-green stereo
views
Orthogonal _____ Creating projection images
Projections along the orthogonal axis
Creating Projection Images
from Different Detection
Channels
SFP (Simulated SFP Creating SFP Projection
Fluorescence Rotation Images
Process) Stereo Running SFP projection
View images as rotation animation

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Creating red-green stereo

views
Filter (Linear) _____ Applying lowpass and
highpass filters to an image
data record
! see Image Tool Dialog Window, 3D Button

Basic Leveling _____ Adapting an interpolation function to
a data set to correct trend curves
Merging Voxel Combining several images or data
x / y / z / ch / sets to a new image or data set
t/λ
Stereo Projections Creating projection images along the
Stereo View orthogonal axis
Creating red-green stereo views
Image _____ Cutting single images or image
Separation sections from a data record

Amplitude Arithmetic With _____ Running Arithmetical Operations
Two Images with Images and/or Masks
Arithmetic With _____ Running Arithmetical Operations
Constant with Images and a Constant
Bit Converter _____ Conversion of 8-bit images to 12-
bit images and vice versa
Brightness and _____ Setting image brightness and
Contrast contrast
Gamma _____ Setting the Contrast Transfer
Correction Function (Gamma Curve)
! see Image Tool Dialog Window, Amplitude Button

Materials Filter _____ Applying low-pass and high-pass filters to
(Linear) images
! see Image Tool Dialog Window, Materials Button

Multicolor Cross _____ Removing cross talk between detection
Talk channels

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Image Tool Dialog Window, 3D Button (optional)

Projections and Creating Projection Images Along a Definable

Animations Icon Projection Axis
Running Projection Images as 3D Rotation
Animation
Creating Red-Green Stereo Views
Creating Projection Images Along a Definable Projection Axis
! In the Projections tab, select one of the three projection types: maximum
projection, average projection, and transparent projection.
! Click on Rescale in the Options field to standardize the intensity values of
the image to the maximum possible intensity values when creating the
projection image. This ensures that the calculated gray values of the
projection image remain within the value range of, for example, 0 to 255 at
8 bits. This allows for brightening dark images.
! If you click on Invert in the Options field, the projection axis is mirrored.
The calculation of the projection image is thus in the opposing direction.
This function makes sense above all in the case of inverse microscopes to
match the calculation of the projection image to the inverse beam path of
laser light.
! The Transparent Factor field is only active if the transparent projection was
selected. You use the slider to vary the transparent factor a. The higher it

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is set, the stronger the intensity values flow from the lower levels in the
image stack into the projection image. In general, this includes more
information in the projection image.
! Move the slider in the Threshold field to define a threshold value. The
intensity values below this value are not taken into account for the creation
of the projection image.
! To set the projection axis, click on the Rotation tab (see below for
description).
! Click on the Show button to obtain a preview of the projection images.
Show starts the calculation and displays the resulting images in the Image
Tool dialog window.
Or
Click on the Apply button to create the projection as a new data record.
Apply starts the calculation, displays the resulting images in the Viewer
window and saves them as a new file in the current experiment.
Note
The three projection types can also be enabled directly using the
corresponding keys (3D Proj / 3D Max / 3D Avg / 3D Trans). If you
activate the function using the button, the projection image will just be a
temporary display on the monitor. You can undo this using the Original
key.
! see Projection of an Image Stack With Variable Projection Axis

! see Maximum Projection of an Image Stack With Variable Projection
Axis
! see Average Projection of an Image Stack With Variable Projection Axis
! see Transparent Projection of an Image Stack With Variable Projection
Axis
Setting the Projection Axis and Running Projection Images as a 3-

dimensional Rotation Animation
! In the Rotation tab, set the desired projection axis by entering a value for
Z, Y and X. With these three values, you rotate the image stack into a
certain position and thus define the angle at which the image stack is
projected. The projection axis here is the viewing angle of the person
viewing the rotated image stack.
You can verify the result in the small preview window in the tab. The
scheme below illustrates the rotation direction of each image level in the
case of positive values for Z, Y and X. In the case of negative values, the
rotation direction is exactly the other way around:
! To make settings for the rotation animation, click on the Animation check
box. The corresponding fields are enabled.
! In the Rotation field, select the animation type: Full turn or 360°, half turn or
180°, quarter turn or 90°, or any other angle.

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! In the Frames field, enter the number of projection images to be created for
the animation. The number of projection images and the animation type
specify the intervals at which the projection images are created. With a half
turn (angle of 180°) and a number of images of, for example, 10, an image
is created in steps of 20° (not 18°), as there are only 9 steps for 10
recorded images.
Note
There is another calculation in the case of the full turn (angle of 360°).
To prevent the first and last projection image from being recorded
under the same angle (360° = 0°), the function calculates one
recorded image more, but without creating the corresponding
projection image. In the case of 10 images, for example, the interval
between the projection images is 36° (not 40°), as 11 recorded
images (i.e. 10 steps) are used in the calculation. However, only 10
projection images are created.
! In the Rotation Axis field, vary the inclination of the rotation axis. Check the
changes in the preview window.
! In the Depth Factor field, enter a value to scale the z-axis of the projection
images.
! Click on the Apply button to start the calculation of the projection, to display
the result image in the Viewer window and to create it as a new file in the
current experiment.
! To start the rotation animation, click on the Movie button.
! see Starting and Ending a Film
Creating Red-Green Stereo Views

On the Stereo View Tab you can create red-green stereo images, so-
called anaglyphic images. Anaglyphic images create a stereoscopic
sense of depth if they are viewed with corresponding 3D glasses (red-
green stereo glasses).
The following settings must be made to create the two stereo images:
! In the Stereo View Options field click on the On check box to activate the
function.
! In the Eye Angle field determine the angle under which the two stereo
images are recorded. For example, if you enter the value 30, a stereo
image is recorded at an angle of 15° each left and right from the projection
axis that was set on the Rotation tab.
! Stereo images can only be created from one detection channel. If you have
an image series that was recorded in several detection channels, select
the desired image data set in the Detection Channel field.
! Click on the Show button to obtain a preview of the stereo images. Show
starts the calculation and displays the resulting images in the Image Tool
dialog window.
Or
Click on the Apply button to create the stereo images as a new data set.
! To display both stereo images in one image in the Viewer window, the
buttons Channel 1, Channel 2, Single and Overlay must be pressed.
! see Viewing Detection Channel 1

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! see Viewing a Single Image

The stereoscopic effect of the anaglyphic image is possible since human
beings use both eyes for viewing. Since the eyes are approximately 6.5
cm (2.56 inch) apart, the brain receives two images from different
perspectives. Objects located at different distances are observed at
different angles. This angular difference is a measure of distance for the
human brain. The difference between the similar images generated by
the two eyes is processed by the brain into one image with spatial depth
impression. Holding a slightly different image in front of each eye can
also simulate these image differences.
In anaglyphic images, the complete stereoscopic image information is

located on one image. The information of the left and right image is
coded through colors. For this reason, glasses with a different color filter
for each eye are needed in viewing these images. This filter separates
the image information again for each eye. The red filter only passes red
light, while completely is absorbing the green light. On the other hand,
the green filter absorbs the red light. Each eye perceives one image; both
are merged in the brain into an overall spatial image.
Icon Orthogonal Creating projection images along the orthogonal

Projection axis
Creating Projection Images from Different
Detection Channels
Creating a Projection Image Along the Orthogonal Axis (z-axis with

horizontal xy-sections and y-axis with vertical xz-sections)
! In the Type field, select one of the three projection types: maximum
! The Direction field determines which single images are used for the
projection.
Select [z] for stereoscopic series, [t] for time series or [l] for wavelength
series to create the projection from all images of a detection channel, such
as from image 1 to image 10 in detection channel 1.
Select [ch] to create the projection from a certain image in all detection
channels, for example from image 1 in detection channel 1 and image 1 in
detection channel 2.
laser light.
selected. The slider is used to vary the transparent factor a. The higher it is
set, the more the intensity values from the lower levels in the image stack
affect the projection image.

Art.No.: 15-9330-033 / Vers.: 01082001

Tool dialog window.
Or
Note
corresponding keys (Fix O. Proj / Fix Max / Fix Avg / Fix Trans). If you
key.
! see Projection of an Image Stack With Invariable Projection Axis

! see Maximum Projection of an Image Stack With Invariable Projection
Axis
! see Average Projection of an Image Stack With Invariable Projection
Axis
! see Transparent Projection of an Image Stack With Invariable Projection
Axis
SFP Icon (Simulated Creating SFP Projection Images

Fluorescence Process) Running SFP Projection Images as 3D
Rotation Animation
Creating a Red-Green Stereo View of
the SFP Projection
Creating an SFP Projection Image
! In the SFP tab in the Light Direction field, you define the projection angle
for the first computing step of the SFP projection. This angle symbolizes
the angle of incidence of the laser beam onto the specimen. Enter one
coordinate each for x and y.
laser light.
! If you click on Filter before Rendering in the Options field, the image stack
is filtered in a lowpass filter in all three spatial directions before the SFP
projection image is calculated. In this way, strong noise can be removed
from the recorded images.
! If you click on Calc. Shadow in the Options field, a shadow is calculated
and displayed for the SFP projection image.
! Move the slider in the Absorption field to vary the absorption coefficient a.

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The higher this is set, the smaller the values calculated in the projection
image. If the coefficient is set as low, many of the pixels in the projection
image reach the maximum value, making it increasingly difficult to
distinguish between the structures.
Tool dialog window.
Or
In the Rotation and Stereo View tabs, you can create a rotation animation
or a stereo view of the SFP projection image.
! see Running Projection Images as a 3-dimensional Rotation

Animation
! see Creating a Red-Green Stereo View
Filter (Linear) Icon Applying low-pass and high-pass filters to images
Filters are used to enhance the quality of an image. As a rule, this means
removing undesirable pixels from the image. The filter function available
here a low-pass filter; is used to suppress static noise in an image that
could be generated by the detector. The principle of a filter consists of
offsetting the value of each pixel in an image with the values of its
neighboring pixel. The filter kernel describes the number and weighting of
the adjacent pixels that come to bear in the calculation of the new pixel.
The kernel used here can be described using Pascal's triangle:
Filter kernel size Pascal triangle Scaling

1 1 1/2
Kernel Size 3 1 2 1 1/4
1 3 1 3 1/8
Kernel Size 5 1 4 6 4 1 1/16
1 5 10 10 5 1 1/32
Kernel Size 7 1 6 15 20 15 6 1 1/64
1 7 21 35 35 21 7 1 1/128
Kernel Size 9 1 8 28 56 70 56 28 8 1 1/256
... ...
If, for example, kernel size 5 is selected, 5 pixels of the original image are
used in calculating 1 pixel in the filtered image. It is always the middle
pixel in the filter kernel that is the pixel to be calculated. In this case, the
gray values of the 5 pixels corresponding to the weighting scale 1-4-6-4-
1, are multiplied by the factors 1/16, 4/16, 6/16, 4/16, 1/16 and together
added to the final value. With respect to the calculation of the pixel C in
the following schematic representation this means that the gray values of
pixels A, B and D, E are included in the calculation of the filtered pixel C'.
C' is calculated as follows: 16x1/16 + 4x4/16 + 8x6/16 + 32x4/16 +
8x1/16 = 1 + 1 + 3 + 8 + 0,5 = 13,5
A B C D E
Gray Values of Pixels A-E in an Original 16 4 8 32 8
Image
Factors of kernel size 5 1/16 4/16 6/16 4/16 1/16

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Gray value of C' in the filtered image 13,5
A calculation of this kind is performed for each pixel in the original image.
At the edge of the image, the filter mask protrudes beyond the image. For
these pixels, the value 0 is set.
! In the Kernel Size field, select a

value for the kernel size.
In the Filter Directions field, set

the dimensions of the filter. Select
the spatial axes to which the filter Intensity profile of an unfiltered image
is to be applied sequentially. One-
dimensional filters are applied,
that is, the filter is calculated only
via one axis at any given time.
! In the Filter Type field, select the
Smooth filter.
The low-pass filter described
above is applied to the image.
This filter type filters the high

Intensity profile of the image after
frequencies out of the image. That
applying the Smooth filter
is, the very distinctive transitions
from low to high intensity values
are reduced.
This filter can be used to remove

noise from an image.
Highpass filter.
After the low-pass filter has been
applied to the image, the values of
the original image are subtracted
from the values of the low-pass-
filtered image. The result
Intensity Profile of the Image After
corresponds to an image that was
Application of the Highpass Filter
processed with a high-pass filter.
This filter type exclusively

represents the clearly defined
transitions from low to high
intensity values in the image.
! In the Filter Type field select the
Sharp filter.
the high-pass-filtered image are
added to the values of the original
Intensity Profile of the Image After
image.
Application of the Sharp Filter
This filter type filters the low
frequencies out of the image. That
is, the very distinctive transitions
from low to high intensity values
are intensified.
This filter is also used to amplify

the noise in an image.

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Use the slider in the Sharp Factor

field to vary the effect of the Sharp
filter. If this factor is set to 0, the
filter is not active.
! The value specified in the Kernel Size field as Cut-off Wavelength, is the
size of the filter measured in pixels. This value is calculated on the basis of
the scan parameters (enlargement of the objective, wavelength of the
excitation light, scan format, electronic zoom factor...) used for each image.
The value specified in the Filter Directions field as Cut-off Wavelength, is
the size of the filter measured in nanometers. For example, with a value of
160 nm all wavelengths lesser than or equal to 160 nm are filtered.
! Click on the Show button to obtain a preview of the filtered images. Show
dialog window.
Or
Click on the Apply button to create the filtered images as a new data
record. Apply starts the calculation, displays the resulting images in the
Viewer window and saves them as a new file in the current experiment.
Image Tool Dialog Window, Basic Button


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Leveling Adapting an interpolation function to

icon a data set to correct trend curves
(e.g., skewed recording of a data
set)
Procedure
This procedure aims at bringing an intensity curve that features a trend
curve into a horizontal position. For this purpose, a suitable interpolation
function (polynomial) is first adapted to the intensity curve. Next, the
distance of the optimal interpolation function to a horizontal intensity
plateau is calculated. The occurring intensity differences are added pixel
by pixel to the original data set. This compensates the trend curve.
! see Adaptation Function for Correcting Trend Curves
The target function to be optimized in determining the coefficients of the

adaptation function is:
Procedure for calculating the adaptation function
! Select an image from the Source Image drop-down list. The drop-down list
contains all the recorded and calculated images of the experiments
displayed in the Experiment Overview viewer window.
! In the Fitting Interpolation Function field select the character of the
adaptation function:
Fitting Interpolation Formula

Function
Linear f(x) = a + bx + cy
Bilinear f(x) = a + bx + cx + dxy
Quadratic f(x) = a + bx + cx + dxy + fx²
Cubic f(x) = a + bx + cx + dxy + fx² + gxy² + hx³ +
iy³
! Select the step size in the Sample Step Width field. The step size sets the
number of support positions for the polynomial. The smaller the step size,
the more time-consuming and precise the calculation of the adaptation
function.
! If the data set should show noise, it is recommended to hide the low
intensity noise signals in the Threshold field by setting a threshold. All
intensity values whose values are below the threshold are not used in the
calculation. This prevents noise signals from being erroneously used as
support positions for the adaptation function.
! Click on the Show button to obtain a preview of the arithmetical operations.
Tool dialog window.
Or
Click on the Apply button to create the calculation operation as a new data
set. Apply starts the calculation, displays the resulting images in the Viewer
Merging Combining several images or data sets to a new image or data

icon set
Voxel tab: Adaptation of different image resolutions and additional

settings for calculating the resulting image

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Field Description of Function

Amplitude The minimum intensity value is shown here. For raw data, this
Image 1/2: value is «0». For scaled data, the actual minimum value is
Min shown here. The minimum is adjustable so that differently
scaled data sets can be adapted to each other. If a data set with
a small interval of intensity values is combined with a data set
featuring a large intensity value range, the first one is shown
darker than the second one. Adapting the minimum values of
both data sets scales them in such a way that equal parts of the
intensity values of both data sets are included in the merged
image.
Amplitude The maximum intensity value is shown here. For raw data, this
Image 1/2: value is «255» with 8-bit data or «4095» with 12-bit data. For
Max scaled data, the actual maximum value is shown here. The
maximum is adjustable so that differently scaled data sets can
be adapted to each other. If a data set with a small interval of
intensity values is combined with a data set featuring a large
intensity value range, the first one is shown darker than the
second one. Adapting the maximum values of both data sets
scales them in such a way that equal parts of the intensity
values of both data sets are included in the merged image.
Result: 8 bit This setting defines the resolution of the merged image.
/ 12 bit An 8-bit resolution can display 256 different intensity levels.
A 12-bit resolution can display 4096 different intensity levels.
Maximum This option takes the brightest pixel each from the two images to
Value be merged to calculate the corresponding pixel of the merged
image.
Average This option calculates the arithmetic mean from the two images
Value to be merged to calculate the corresponding pixel of the merged
image.
Note
If two strongly different images are calculated to a merged

image by using the average method, the black image
background causes a reduction of the image intensity!
Tab x, y, z, t, la: Definition of the stereoscopic arrangement of the

output images in the resulting image before combining

Image 1/2: The position in x/y/z (t/λ) of the output image within the merged
Shift image is set here. This value is typically «0». A value of > 0 is
useful for example, if a reduction of one output image is to be
shown at a certain position of the other output image. This
function is also useful for lateral image series, such as those
found in biomapping.
! see Example for Shifting an Image
Append The second data set is now placed next to the first data set. The
check box direction of the placement depends on whether the check box on
the x, y or z tab was selected. Selecting this function in all the
different tabs for x, y and z shifts the second output image either
in only one dimension or in several dimensions.
Result: Here you can indicate which pixel represents the end of the
Element merged image.
number Definition background: Absolute lengths are used to determine

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the dimensions of the merged image. If the two data sets to be

merged feature the same linear extension but a different number
of pixels (due to a different scan format), it must be decided
whether the number of pixels or the resulting total length is to be
decisive for the merged image.
Result: The program uses the following procedure upon selecting «Best
Best Fit Fit»:
The width of a single pixel is calculated for both images: µm/pixel.
If the difference of this size between the two images is smaller
than a factor of 2, this procedure uses the larger image resolution
(i.e., the smaller µm/pixel value) for the resulting image. If the
deviation of the image resolution between the two output images
is larger than a factor of 2, the smaller image resolution (i.e., the
larger µm/pixel value) is used for calculating the resulting image.
The number of pixels of the resulting image (Element Number)
divided by the total length results in the resolution of the resulting
image.
Definition background: Absolute lengths are used to determine
the dimensions of the merged image. If the two data sets to be
merged feature the same linear extension but a different number
of pixels (due to a different scan format), it must be decided
whether the number of pixels or the resulting total length is to be
decisive for the merged image.
Tab ch: Definition of the combination of images with different

numbers of detection channels

Image 1: If two images or data sets with different numbers of detection
Start channels are to be merged, this option can be used to define the
channel number in the merged image at which the channels of the
output image 1 should be inserted.
! see Example for merging images with different channel
numbers
Image 2: If two images or data sets with different numbers of detection
Start channels are to be merged, this option can be used to define the
channel number in the merged image at which the channels of the
output image 2 should be inserted.
! see Example for merging images with different channel
numbers
Check This function distributes the two output images to different
box: channels of the merged image.
Append
Stereo icon Creating projection images along the orthogonal axis

Creating red-green stereo views
Creating projection images along the orthogonal axis (z-axis for

horizontal xy-sections and y-axis for vertical xz-section)
! In the Type field, select one of the three projection types: maximum
! The Direction field determines which single images are used for the
projection.
Select [z] for stereoscopic series, [t] for time series or [l] for wavelength
series to create the projection from all images of a detection channel, such

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as from image 1 to image 10 in detection channel 1.

Select [ch] to create the projection from a certain image in all detection
channels, for example from image 1 in detection channel 1 and image 1 in
detection channel 2.
laser light.
selected. The slider is used to vary the transparent factor a. The higher it is
set, the more the intensity values from the lower levels in the image stack
affect the projection image.
Tool dialog window.
Or
Note
corresponding keys (Fix O. Proj / Fix Max / Fix Avg / Fix Trans). If you
key.
! see Projection of an Image Stack with Invariable Projection Axis

! see Maximum Projection of an Image Stack with Invariable Projection
Axis
! see Average Projection of an Image Stack with Invariable Projection Axis
! see Transparent Projection of an Image Stack with Invariable Projection
Axis
On the Stereo View Tab you can create red-green stereo images, so-
called anaglyphic images. Anaglyphic images create a stereoscopic
sense of depth if they are viewed with corresponding 3D glasses (red-
green stereo glasses).
The following settings must be made to create the two stereo images:
! In the Stereo View Options field click on the On check box to activate the
function.
! In the Eye Angle field determine the angle under which the two stereo
images are recorded. For example, if you enter the value 30, a stereo

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image is recorded at an angle of 15° each left and right from the projection
axis that was set on the Rotation tab.
! Stereo images can only be created from one detection channel. If you have
an image series that was recorded in several detection channels, select
the desired image data set in the Detection Channel field.
! Click on the Show button to obtain a preview of the stereo images. Show
dialog window.
Or
Click on the Apply button to create the stereo images as a new data set.
! To display both stereo images in one image in the Viewer window, the
buttons Channel 1, Channel 2, Single and Overlay must be pressed.
! see Viewing a Single Image
The stereoscopic effect of the anaglyphic image is possible since human
beings use both eyes for viewing. Since the eyes are approximately 6.5
cm (2.56 inch) apart, the brain receives two images from different
perspectives. Objects located at different distances are observed at
different angles. This angular difference is a measure of distance for the
human brain. The difference between the similar images generated by
the two eyes is processed by the brain into one image with spatial depth
impression. Holding a slightly different image in front of each eye can
also simulate these image differences.
In anaglyphic images, the complete stereoscopic image information is

located on one image. The information of the left and right image is
coded through colors. For this reason, glasses with a different color filter
for each eye are needed in viewing these images. This filter separates
the image information again for each eye. The red filter only passes red
light, while completely is absorbing the green light. On the other hand,
the green filter absorbs the red light. Each eye perceives one image; both
are merged in the brain into an overall spatial image.
Image Separation Separating single images or image segments from a

icon data set
Here you perform the settings to separate a segment from an image or

complete images from an image stack in one or several detection
channels.
! The dimensions x, y and z and the detection channel can freely be
combined in the Dimension Selection field:
Field Function description

Dimension The detection channels of the image or the image series are
Selection ch indicated here. Select the detection channels from which the
image segment or the individual images are to be separated.
Dimension The number of single images of an image series is indicated
Selection z here. Enter the number of images to be separated in the Start
z and End z entry fields. Use the R (Reset) button to undo the

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entries.
Dimension The number of pixels in y that is dependent upon the scan
Selection y format is indicated here. Enter the number of pixels to be
separated in the Start y and End y entry fields. Use the R
(Reset) button to undo the entries.
Dimension The number of pixels in x that is dependent upon the scan
Selection x format is indicated here. Enter the number of pixels to be
separated in the Start x and End x entry fields. Use the R
(Reset) button to undo the entries.
Step z / y / x Here you can enter an interval within a dimension. For
example, if you enter 2 in the Step z entry field, only every 2nd
image is separated.
! If you want to separate all images of a detection channel from an image

series, click in the ch entry field on the channel to be separated without
changing the values in the entry fields of the other dimensions.
! If you want to separate complete single images from an image series,
enter the corresponding numbers in the Start z and End z entry fields
without changing the values in the entry fields of the other dimensions.
! If you want to separate a segment in all images of an image series, define
the size of the segment in the Start x, End x and Start y, End y entry fields
without changing the values in the entry fields for z.
Image Tool Dialog Window, Amplitude Button


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Arithmetic With Two Running Arithmetical Operations with Images

Images Icon and/or Masks
Procedure
! Select two images from the Source Image 1 and Source Image 2 drop-
down lists. The two drop-down lists contain all the recorded and calculated
images of the experiments displayed in the Experiment Overview viewer
window.
! Select the desired arithmetical operation in the Operation field.
Note
A requirement for running arithmetical operations is equal dimensions or

a 1/n relationship of the two data records. Equal dimensions means that
two images must have the same format in x and y, and that two image
stacks must have the same number of single images (z). The 1/n
relationship exists when, for example, 1 single image is offset against
each (nth) single image of an image stack.
The following arithmetical functions can be run with two images

and/or masks:
Operation Result
Addition Adds the intensity values of the two selected images according
to the formula:
Pixel Intensity Result = Pixel Intensity Source Image 1 + Pixel Intensity
Source Image 2
Subtraction Subtracts the intensity values of the two selected images
according to the formula:
Pixel Intensity Result = Pixel Intensity Source Image 1 - Pixel Intensity
Source Image 2
Multiplication Multiplies the intensity values of the two selected images
Pixel Intensity Result = Pixel Intensity Source Image 1 x Pixel Intensity
Source Image 2
Division Divides the intensity values of the two selected images
Pixel Intensity Result = Pixel Intensity Source Image 1 / Pixel Intensity
Source Image 2
Min Selects for each pixel the minimum value of the two selected
images according to the formula:
Pixel Intensity Result = MIN (Pixel Intensity Source Image 1; Pixel
Intensity Source Image 2)
Max Selects for each pixel the maximum value of the two selected
Pixel Intensity Result = MAX (Pixel Intensity Source Image 1; Pixel
Average Calculates for each pixel the average of the two selected
Pixel Intensity Result = 1/2 x (Pixel Intensity Source Image 1 + Pixel
AND The binary representations of the pixels of the two selected

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images are linked with the logical operator AND. That means
that pixels in the result image are given the value 1 when the
value 1 is present in both of the pixels; otherwise 0 is entered.
You achieve meaningful results using this Boolean operator
when you link an image with a binary mask. The pixel is
displayed in the result image at the point where the pixels of the
image meet the pixels of the mask with the value 1. The areas
defined by the mask are cut out of the image.
OR The binary representations of the pixels of the two selected
images are linked with the logical operator OR. That means that
pixels in the result image are given the value 1 when the value
1 is present in at least one of the two pixels; otherwise 0 is
entered.
You achieve meaningful results using this Boolean operator:
1. When you link two binary masks. The result image is a
combination of the two masks. 2. When you link an image with
a binary mask. The result image is an image in which the mask
appears. 3. When you link two images. In contrast to the
overlay image, pure intensity values are overlaid here. An RGB
color addition does NOT take place here.
XOR The binary representations of the pixels of the two selected
images are linked with the logical operator XOR. That means
two pixels have different values; otherwise 0 is entered.
You achieve meaningful results using this Boolean operator
when you create two binary masks of images and link them.
The pixel is displayed in the result image at the point where the
two images differ.
! In the Arithmetic Type field, select whether raw data or scale data is to be
used for the arithmetical operations.
! If you have selected Raw Data, the values standardized to the interval
[0..255] for 8 bits or [0..4095] for 12 bits are used for the arithmetical
operations. As the calculation result can produce values extending beyond
the interval, the following three methods are available to readapt them to
the interval:
Raw Data Result

Options
Cut at Values less than 0 are set to 0. All values greater than 255 for 8-
Maximum bit images or 4095 for 12 bit images are set to the value 255 or
4095. This "concatenates" all the values lying outside the
interval.
Absolute The amounts of the result data are used. This turns negative
value results into positive results.
Rescale The extreme values are determined. The minimum value is
assigned to the 0. The maximum value for 8-bit images is
assigned to the value 255, for 12-bit images to the value 4095.

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! If you have selected Scaled Data, the world coordinates, that is, the actual
and non-standardized values, are used for the arithmetical operations. The
standardization of the results data into the intensity interval [0..255] and/or
[0..4095] is only used for the image display functions for color look-up; in
contrast, the actual result values are used for the quantification functions.
! If you select 12 Bit Output in the Output Option field, each pixel in the
result image is coded with 12 bits, i.e. the result values are saved in a
value range from [0..4095]. In the case of scaled data, the intermediate
values are interpolated.
Tool dialog window.
Or
Arithmetic With Constant Running Arithmetical Operations with Images

Icon and a Constant
Procedure
! Set the desired value of the constants in the Constant Value field.
! Select the desired arithmetical image operation in the Operation field.
The following arithmetical functions can be run with one image and
a constant:
Operation Result
Addition Adds the constants pixel by pixel to the intensity values of the
selected image according to the formula:
Pixel Intensity = Pixel Intensity
Result + Constants
Source Image
Subtraction Subtracts the constants pixel by pixel from the intensity values
of the selected image according to the formula:
Result - Constants
Source Image
Multiplication Multiplies the constants pixel by pixel by the selected image

Result x Constants
Source Image
Division Divides the selected image by the constants according to the

formula:
Result / Constants
Source Image
Min Selects for each pixel the minimum value of the selected image
and constants according to the formula:
Pixel Intensity = MIN (Pixel Intensity
Result ; Constants)
Source Image
Max Selects for each pixel the maximum value of the selected image
and constants according to the formula:
Pixel Intensity = MAX (Pixel Intensity
Result ; Constants)
Source Image
Average Calculates for each pixel the average of the selected image and
constants according to the formula:
Pixel Intensity = 1/2 x (Pixel Intensity
Result + Constants)
Source Image
AND The binary representations of the pixels of the constants and

the image are linked with the logical operator AND. That means
value 1 is present in both of the pixels; otherwise 0 is entered.

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OR The binary representations of the pixels of the constants and

the image are linked with the logical operator OR. That means
value 1 is present in at least one of the two pixels; otherwise 0
is entered.
XOR The binary representations of the pixels of the constants and
the image are linked with the logical operator XOR. That means
two pixels have different values; otherwise 0 is entered.
! In the Arithmetic Type field, select whether raw data or scale data is to be
used for the arithmetical operations.
! If you have selected Raw Data, the values standardized to the interval
[0..255] for 8 bits or [0..4095] for 12 bits are used for the arithmetical
operations. As the calculation result can produce values extending beyond
the interval, the following three methods are available to readapt them to
the interval:
Raw Data Result

Options
Cut at Values less than 0 are set to 0. All values greater than 255 for 8-
Maximum bit images or 4095 for 12 bit images are set to the value 255 or
4095. This "concatenates" all the values lying outside the
interval.
Absolute The amounts of the result data are used. This turns negative
value results into positive results.
Rescale The extreme values are determined. The minimum value is
assigned to the 0. The maximum value for 8-bit images is
assigned to the value 255, for 12-bit images to the value 4095.
! If you have selected Scaled Data, the world coordinates, that is, the actual
and non-standardized values, are used for the arithmetical operations. The
standardization of the results data into the intensity interval [0..255] and/or
[0..4095] is only used for the image display functions for color look-up; in
contrast, the actual result values are used for the quantification functions.
! If you select 12 Bit Output in the Output Option field, each pixel in the
result image is coded with 12 bits, i.e. the result values are saved in a
value range from [0..4095]. In the case of scaled data, the intermediate
values are interpolated.
Tool dialog window.
Or
Bit Converter Conversion of 8-Bit Image Resolution to 12-Bit Image

Icon Resolution and Vice Versa
Procedure
! In the New Resolution field, select the image resolution into which you
want to convert the selected image.

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! Click on the Show button to obtain a preview of the converted image.

Tool dialog window.
Or
Click on the Apply button to create the converted image as a new data
Brightness & Contrast Icon Setting image brightness and contrast
Procedure
! For multichannel images, select the All Channels check box to make all
changes for all channels at one time and to the same extent.
! For images that do not use the entire dynamic range, select the Linear
Model check box. If the check box has been clicked, there is a linear
relationship between the intensity in the output image and the result
image. Whether an image uses the entire dynamic range available can be
seen by creating a histogram of the image. If the histogram does not cover
the entire intensity range (see Min and Max), the dynamic range is NOT
being used in full.
! see Calculating a Histogram
If you have images in which you wish to highlight the medium intensities
more strongly, select the non-linear model (Linear Model check box NOT
enabled).
! In the Options field, use the sliders to change the brightness and/or image
contrast.
Options Result
Slider This slider adjusts the brightness of the result image. If the slider
Brightness is exactly in the center, the output image and result image have
exactly the same level of brightness. If the slider is moved from
the center to the right, the result image becomes brighter than the
output image. If the slider is moved from the center to the left, the
result image becomes darker than the output image.
Slider This slider adjusts the contrast of the result image. If the slider is
Contrast exactly in the center, the output image and result image have
exactly the same level of contrast. If the slider is moved from the
center to the right, the result image has greater contrast than the
output image. If the slider is moved from the center to the left, the
result image has less contrast than the output image.
! Click on the Show button to obtain a preview of the calculation. Show

dialog window.
Or
Click on the Apply button to create the calculation as a new data record.
Gamma Correction Setting the Contrast Transfer Function (Gamma

Icon Curve)
The contrast transfer function (gamma curve) can be used to influence

the conversion of the output image contrast to the result image contrast
in a non-linear manner.

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Procedure
! For multichannel images, select the All Channels check box to make all
changes for all channels at one time and to the same extent.
! Change the form of the contrast transfer function by adjusting the slider.
! Click on the Show button to obtain a preview of the calculation. Show
dialog window.
Or
Click on the Apply button to create the calculation as a new data record.
Viewing detection channels
Viewing Detection Channel 1-8
Function
Clicking the Channel 1/2/3/4/5/6/7/8 button displays the image data that
has been recorded in detection channel 1-8 in the Viewer window. You
can assign any of several other color look-up tables to the detection
channel. This setting can be changed both in the result image and while
the image is being recorded. To do so, open the Select Look-up Tables
dialog window. There are two ways of opening the dialog window.
! Click the Select Look-up Tables button.

! Hold the mouse pointer over any position in the Viewer window.
Click the right mouse button. Click on the LUT item in the context menu
that appears.
The color bars for the active detection channels appear in the Viewer
window.
Double-click the corresponding color bar.
! see Selecting Color Look-Up Tables (LUT)
Note
This method of assigning color look-up tables only influences the

currently displayed image. As soon as you start a new scan, the color
look-up tables configured in the Beam Path Setting dialog window are
applied again.

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Zooming Image(s) in the Viewer Window
Function
Clicking the Display button opens a drop-down list that you can use to set
the graphical zoom. This enlarges or reduces the image displayed in the
Viewer window:
n-1 Displaying n pixel as 1 pixel in the resulting image reduces the

image.
Automatic The image is displayed in the original image format.
1-n Displaying 1 pixel as n pixel in the resulting image enlarges the
image.
Note
graphic zoom, the 3D zoom and the electronic zoom.

Selecting Color Maps (LUT)
Function
Use the Look-up Tables button to open a dialog window, which you can
use to assign color look-up tables to the detection channels. The color
look-up tables can be configured both in the result image and while the
image is being recorded:
! Click the detection channel to which you want to assign a new color look-
up table in the Select Channel field.
! Select the desired color look-up table in the Select LUT field.
! Click Apply to check the results in the Viewer window.
It is also possible to open the Select LUT's dialog window from the
Viewer window.
! Hold the mouse pointer over any position in the Viewer window. Then push
the right mouse button. Click on the LUT item in the context menu that
appears.
! The color bars for the active detection channels appear in the Viewer
window to the right of the image window. Double-click one of the color
bars.

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Loading a second color look-up table

If you hold the mouse pointer over a color bar, grab points appear at the
top and bottom of the color bar. You can use these grab points to limit
the current color look-up table to a specific intensity range and to load a
second color look-up table:
! Drag the top grab point down or the bottom grab point up.
! Double-click the area above or below the corresponding grab point.
! The Select LUT's dialog window opens so that you can select a second
color look-up table.
! The upper and lower intensity ranges are represented in the colors of the
second color look-up table.
Note
This method of assigning color look-up tables only influences the

currently displayed image. As soon as you start a new scan, the color
look-up tables configured in the Beam Path Setting dialog window are
applied again.
Generally the selection of a suitable color look-up table for a specific use
depends on the user's own judgment and needs. Experience shows
however that certain color look-up tables are particularly useful for
specific uses.
Color Map Application

Green This is commonly used for recording specimens that have been
marked with FITC, Cy2, DTAF or other similar fluorochromes,
which emit within the green spectral range.
Red This is commonly used for recording specimens that have been
marked with TRITC, Texas Red, Cy3, Rodamine or other similar
fluorochromes, which emit within the red spectral range.
Blue This is commonly used for recording specimens that have been
marked with UV fluorochromes such as DAPI or Hoechst or other
similar fluorochromes, which emit within the blue spectral range.
Gray This is commonly used for displaying transmission recordings.
P. Color 1 This is especially suited for displaying 12 bit images.
P. Color 2, This is commonly used for recording specimens that have been
3, 4, 5, 6 marked with pH-sensitive or ion-sensitive fluorochromes and for
displaying time series.
Geo These are recommended for generating topographical images,
(Land), i.e. for mapping surface structures. Geo Land & Sea is especially
Geo (Sea) useful for visualizing depths and graduations.
Geo (Land
& Sea)
R&B
Glow, These are recommended for optimizing image contrast (the offset
Glow and gain of the detectors). Glow Over highlights in blue
(Over), intensities at the upper end of the table while Glow Under
Glow represents in green intensities at the lower end of the table. Glow
(Under), Over and Under is a combination of these two color tables.
Glow (Over
& Under)

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Cyan, These are recommended for printing images. The CMY(K) colors
Magenta, are generally used for the printer system color output. RGB
Yellow colors such as the other color look-up tables used here are
applied generally for color representation on monitors. Because
these two color systems vary greatly, the colors in the CMY(K)
representation can differ strongly from those used in the monitor
representation.
All color look-up tables are also available with inverse color flow,
meaning high intensities are represented as dark and low intensities light.
Viewing the Single image
Function
Click the Single button to view only one detection channel or several
detection channels in only one image in the Viewer window. The
following display modes are available depending on the other options
you enable simultaneously.
Button Combination Display

Single + Channel 1 / 2 / Only the selected detection channel is displayed;
3/4/5 for an image series, the first image of the selected
detection channel is displayed.
Single + Gallery + All individual images in an image series are
Channel 1 / 2 / 3 / 4 / 5 displayed for the selected detection channel only.
Single + Overlay + A single overlay image is generated from all
Channel 1 / 2 / 3 / 4 / 5 selected detection channels; for an image series,
the first image of each selected detection channel is
used.
Single + Gallery + Based on the number of individual images in an
Overlay + Channel 1 / 2 image series, overlay images are generated from
/3/4/5 all selected detection channels.
Note
The Single and Tiled buttons cannot be enabled simultaneously because

they carry out opposing functions.

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Viewing a Multiple image
Function
If you click the Tiled button, the detection channels are displayed
separately in the Viewer window. The following display modes are
available depending on the other options you enable simultaneously.

Tiled + Channel 1 / 2 / 3 All selected detection channels are displayed
/4/5 separately; in the case of image series, the first
image in each is applied.
Tiled + Gallery + All individual images in an image series are
Channel 1 / 2 / 3 / 4 / 5 displayed for all selected detection channels.
Tiled + Overlay + All selected detection channels are displayed both
Channel 1 / 2 / 3 / 4 / 5 separately and, in an overlay image, together. In the
case of image series, the first image in each is
applied.
Tiled + Gallery + All individual images of an image series are
Overlay + Channel 1 / 2 displayed both separately for all selected detection
/3/4/5 channels and, in overlay images, together.
Note
The Tiled and Single buttons cannot be enabled simultaneously because

they carry out opposing functions.
Viewing an Overlay Image
Function
Clicking the Overlay button displays all selected detection channels
together in an overlay image in the Viewer window. The following display
modes are available depending on the other options you enable
simultaneously.

Single + Overlay + A single overlay image is generated from all
Channel 1 / 2 / 3 / 4 / 5 selected detection channels; for an image series,
the first image of each selected detection channel is
used.
Gallery + Single + Based on the number of individual images in an
Overlay + Channel 1 / 2 image series, overlay images are generated from
/3/4/5 all selected detection channels.
Tiled + Overlay + All selected detection channels are shown

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Channel 1 / 2 / 3 / 4 / 5 separately as well as together in an overlay image.

In an image series, the first image is used.
Tiled + Gallery + All individual images of an image series are
Overlay + Channel 1 / 2 displayed both separately for all selected detection
/3/4/5 channels and, in overlay images, together.
Use the Viewer Options dialog window to select from three different
methods of color mixing for generating an overlay image:
! From the View menu, select Viewer Options.

! Click on the icon Overlay.
! see Viewer Options dialog window, icon Overlay
Viewing Image Series
Viewing the First Image of a Series
Function
You can view the individual images of an image series as a film
sequence. Click First to jump to the first image in the series.
Note
If the Gallery button is enabled, the First and Last, Next and Previous
and Play/Stop buttons are disabled and displayed in gray. The Gallery
button is used to show all of the individual images of an image series and
does not allow film sequence mode.
Display the Next Image in a Series
Function
sequence. Click Next to jump to the next image in the series.

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Note
If the Gallery button is enabled, the Next and Previous, First and Last
Displaying the Previous Image in a Series
Function
sequence. Click Previous to jump to the previous image in the series.
Note
If the Gallery button is enabled, the Next and Previous, First and Last
Viewing the Last Image of a Series
Function
sequence. Click on the Last button to jump to the last image in the series.
Note
If the Gallery button is enabled, the First and Last, Next and Previous

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Displaying an Image From a Series
Function
When you click on the Selection button, a dialog window opens which
lets you select any image of a series and display it in the Viewer window.
The number of individual images for the series is displayed from 1 to n,
as well as in an entry field the number of the currently displayed image.
! Use the mouse to move the slide of the scale. The corresponding image is
displayed immediately.
! Enter the number of the desired image into the entry field, and click on
Apply.
Starting and Ending a Film
Function
You can view the individual, recorded images of an image series as a
film sequence. Use the Play/Stop button to start and stop the film. The
film speed, that is the number of single images per time unit, is variable
and can be set in the Viewer Options dialog window:

! Click the Display icon and then on the Movie tab.
! see Viewer Options dialog window, Display icon
Note
If the Gallery button is enabled, the Play/Stop, First and Last, Next and
Previous buttons are disabled and displayed in gray. The Gallery button
is used to show all of the individual images of an image series and does
not allow film sequence mode.

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Viewing the Series image
Function
If you click the Gallery button, all of the individual images in an image
series are displayed in the Viewer window. The following display modes
are available depending on the other options you enable simultaneously.

Gallery + Single + All individual images in an image series are
Channel 1 / 2 / 3 / 4 / 5 displayed for the selected detection channel only.
Gallery + Single + Based on the number of individual images in an
Overlay + Channel 1 / 2 / image series, overlay images are generated from
3/4/5 all selected detection channels.
Gallery + Tiled + Channel All individual images in an image series are
1/2/3/4/5 displayed for all selected detection channels.
Gallery + Tiled + Overlay All individual images of an image series are
+ Channel 1 / 2 / 3 / 4 / 5 displayed both separately for all selected detection
channels and, in overlay images, together.

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Projections
Principles and Types of Projections
(2a) Projection with an invariable projection axis

(2b) Projection with a variable projection axis
Functional Principles of Projections
Biological specimens are recorded with a series of images. Each image is
taken from a particular level in the specimen. The result is a three dimensional
recording that contains the entire volume of a specimen. Each optical section
(each individual image) depicts only the information from a single level in the
specimen. Therefore, structures that extend throughout the entire specimen will
be partially represented in several images. Projections are necessary to view
these structures in their entirety. Using projection algorithms, it becomes
possible to select the relevant information from many images and to depict this
information in a single two-dimensional image. This method can be used to
reconstruct connected structures.
Accordingly, a series of images is the foundation of any projection. The series

may consist of horizontal xy-sections or vertical xz-sections. In order to best
understand the functionality of a projection, it is practical to think of an image
series as a stack of individual images. In turn, each individual image consists of
individual laser sampling points. Furthermore, the individual sampling points or
Voxels (1), (the intensity values measured in each individual image) lie on top
of each other. During creation of a projection, the sampling points of the
individual images, which lie on top of each other along the projection axis (2a,
2b), are scanned through all of the optical sections. Then, either the intensity
value that fulfills the selection criteria or another calculated value is depicted in
a two-dimensional image (4a, 4b) as a representative of all intensity values

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within the column. This is repeated for each column of sampling points (3a, 3b).
Projections with invariable or variable projection axes

There are two main groups of projection types available in the Leica Confocal
Software. These are distinguished by the type of projection axis. There are
projections with invariable axes as well as projections with variable axes. The
first type is orthogonal projections. The projection axis is always the optical axis
that runs vertical to the stack of images (the z-axis with horizontal xy-cuts and
the y-axis with vertical xz-cuts). In contrast, the projection axis for the second
type may be freely selected. With the exception of the SFP projection, the
same three projection types are available in both of the two main groups.
The Projection Types
Projection Type Invariable Projection Variable Projection

Axis Axis
Maximum Projection
Average Projection
Transparent
Projection
SFP Projection __________
Activate the projection type either with the corresponding button or in the
Image Tool dialog window. If you activate the function using the button,
the projection image will just be a temporary display on the monitor.
When activated through the dialog window, the projection image will be
stored as a new file in the current experiment:
! The projections with invariable projection axes are located in the Image
Tool dialog window / 3D button / Orthogonal Projection icon.
! The projections with variable projection axes are located in the Image Tool
dialog window / 3D button / Projections and Animations icon.
! The SFP projection is located in the Image Tool dialog window / 3D button
/ SFP icon.
Maximum Projection
During maximum projection, we assume that the maximum intensity
values are the relevant information for the reconstruction of a structure.
For this reason, the maximum value is found in each column of sampling
points and is displayed in the two dimensional projection image as
representative of the entire column:
Ip = Max(Vn)
Whereby Ip is the pixel in the projection image and Vn is the investigated
Voxel
Average Projection
During average projection, every intensity value with equal weighting
flows into the projection image. For this reason, during average
projection the arithmetic average of all intensity values is calculated in
each column of sampling points. This value is displayed in the two
dimensional projection image as representative of the entire column:

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whereby IP is the pixel in the projection image, Vn is the investigated

Voxel, and N is the total number of Voxel
Transparent Projection
During transparent projection, all intensity values should be included in
the projection image as well. In contrast to average projection, the
intensity values from the various individual images are weighted
differently. The intensity values from the lower images in the image stack
are weighted less than the intensity values from the upper images. For
this reason, during transparent projection the weighted mean of all
intensity values is calculated in each column of sampling points. This
value is displayed in the two dimensional projection image as
representative of the entire column:
Whereby In is the pixel in the projection image, Tn = 1 - (Vn / Vmax) is

the standardization, a is the variable transparent factor, In-1 is the value
of the previously calculated Voxels, and Vn is the currently investigated
Voxel
Calculation of the projection image begins with the lowest and ends with
the highest individual image in the image stack. Weighting of the intensity
values is based on two factors.
The first factor, Tn, is calculated from the relationship of the respective
intensity value to the maximum possible intensity (standardization). This
factor ensures that an image's gray value range from 0 to 255 at 8 bit, for
example, will not be exceeded.
The second (user adjustable) transparent factor a (0 < a < 1) determines
the weighting of the previously measured intensity value. The higher the
transparent factor is adjusted, the more the intensity values from the
lower levels in the image stack will be included in the projection image.
SFP Projection (Simulated Fluorescence Process), mathematical

simulation of a fluorescence process
This projection type uses arithmetic operations to simulate in a specimen
the process of absorbing laser light and emitting fluorescent light. This
procedure is common in fluorescence microscopy. It consists of two
arithmetic steps.
The first step simulates the laser beam (1), which penetrates the sample

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and is thereby weakened. We assume a lateral light source striking the

Voxel of the image stack. The weakening of the incoming beam while
traversing the image stack is represented by the following expression:
Whereby In+1 is the remaining intensity of the incoming light after

absorption in the nth Voxel, In is the intensity of the incoming light in the
nth Voxel, a is the absorption coefficient of the laser light in the sample,
Vn is the intensity in the Voxel, and Vmax is the maximum Voxel
intensity. The quotient Vn / Vmax corresponds to the standardization.
For each Voxel, the illumination strength VIn = In x Vn is calculated using

the calculated intensities (In). This information is temporarily saved in a
table (In = intensity of the incoming light in Voxel n, Vn = intensity of the
Voxel n). This calculation is completed in one step for the entire image
stack (for all Voxel in the direction of the incoming beam). The result is a
table of illumination strengths.
In the second step, the established illumination strengths are used to

calculate the emitted fluorescence (2). The recursive formula is used to
calculate the intensity of the pixel in the projection image as
representative of the Voxel in the observer's viewing direction.
In addition, a is the absorption coefficient of the fluorescent light in the

sample and VIn is the illumination strength of the nth Voxel, calculated in
the first step.
Projecting an Image Stack with an Invariable

Projection Axis
Function
Using the button Fix. O. Proj., start the projection type that was used last
or which was selected in the Viewer Options dialog window/ Projections
icon/ Projections tab. For this function, the projection axis is always the
orthogonal axis (z-axis for horizontal xy-sections, and y-axis for vertical
xz-sections).
The basis for a projection is an image stack, i.e. a series of horizontal xy-
sections or vertical xz-sections. When a projection is generated, the
sampling points of the individual images—superimposed along the
projection axis—are examined throughout all optical sections.
From each of these columns comprised of sampling points, depending on
the projection type, the maximum intensity value or the arithmetical
average or, the weighted average of all intensity values, is calculated and
displayed in a two-dimensional projection image representing the entire
column.

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! Click the Fix. O. Proj. Button. A maximum, average, or transparent

projection of the current image stack is created and displayed in the Viewer
window.
! You can return to the original image anytime by clicking on the Original
button.
The Viewer Options dialog window allows you to indicate a threshold and
to scale the intensity value range for the image.
! see Viewer Options Dialog Window, Projections Icon
Note
You can activate a projection using either the Fix O. Proj. Button or the
stored as a new file in the current experiment.

Projecting an Image Stack with a Variable

Projection Axis (optional)
Function
The 3D Proj. Button starts the project type last used or selected in the
Image Tools dialog window/ Projections and Animations icon/ Projections
tab. The projection axis is freely variable.
sampling points of the individual images superimposed along the
projection axis;are examined throughout all optical sections. From each
of these columns comprised of sampling points, depending on the
projection type, the maximum intensity value or the arithmetical average
or, the weighted average of all intensity values, is calculated and
displayed in a two-dimensional projection image representing the entire
column.
! Click the 3D Proj. Button. A maximum, average, or transparent projection

of the current image stack is created and displayed in the Viewer window.
! You can now change the projection axis by clicking in the image in the
Viewer window and holding the left mouse button pressed.
! Broken support lines are displayed that encloses the symbolically

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displayed image stack. Turn the image stack to the desired new position by
moving the mouse pointer while holding down the left mouse button.
! Upon releasing the left mouse button, the new projection will be calculated
and displayed. The projection axis is the viewing direction of the user to the
newly positioned image stack.
! Use the Zoom 3D View button to enlarge or reduce the projection.
button.
The Viewer Options dialog window allows you to indicate a threshold and
to scale the intensity value range for the image.
Note
You can activate a projection by either using the 3D Proj. Button or the
stored as a new file in the current experiment.

Maximum Projection of an Image Stack with

Invariable Projection Axis
Function
Using the button Fix. Max. Button starts a maximum projection. For this
function, the projection axis is always the orthogonal axis (z-axis for
horizontal xy-sections, and y-axis for vertical xz-sections).
projection axis are examined throughout all optical sections. From each
of these columns of sampling points, the maximum intensity value is
displayed in the two-dimensional projection image as the representative
of all intensity values within the column.
! Click the Fix. Max. Button. A maximum projection of the current image
stack is created and displayed in the Viewer window.
button.

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In the Viewer Options dialog window, you can also adjust a threshold and
scale the intensity value range present in the image.
Note
A maximum projection can be activated either by using the Fix. Max.

Button or in the Image Tool dialog window via 3D button, Orthogonal
Projection icon. If you activate the function using the button, the
projection image will just be a temporary display on the monitor. When
activated through the dialog window, the projection image will be stored
as a new file in the current experiment.

Note
Note the difference between a maximum projection and a topographical

image, which is generated from intensity maximums. In a maximum
projection, the intensity maximums are assigned color values directly. In
a topographical image based on intensity maximums, the intensity
maximums are assigned real z positions of the individual sampling points
first and then are color-coded.
Maximum Projection of an Image Stack with

Function
The 3D Max. Button starts a maximum projection. The projection axis is
freely variable.
of these columns of sampling points, the maximum intensity value is
displayed in the two-dimensional projection image as the representative
of all intensity values within the column.

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! Click on the 3D Max. Button. A maximum projection of the current image

! If you release the left mouse button, a new maximum projection is
calculated and displayed. The projection axis is the viewing direction of the
user to the newly positioned image stack.
button.
Note
A maximum projection can be activated either with the 3D Max. Button or

in the Image Tool dialog window with the 3D button and the Projections
and Animations icon. If you activate the function using the button, the

Note
Note the difference between a maximum projection and a topographical

image, which is generated from intensity maximums. In a maximum
projection, the intensity maximums are assigned color values directly. In
a topographical image based on intensity maximums, the intensity
maximums are assigned real z positions of the individual sampling points
first and then are color-coded.

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Average Projection of an Image Stack With Variable

Projection Axis (optional)
Function
Use the button 3D Avg. to start an average projection. The projection
axis is freely variable.
of these columns of sampling points, the arithmetic average is calculated
from all intensity values and displayed in the two-dimensional projection
image as the representative of the complete column.
! Click on the 3D Avg. button to create an average projection of the current

stack of images. The projection is displayed in the Viewer window.
displayed image stack. Turn the image stack to the desired new position
by moving the mouse pointer while holding down the left mouse button.
! After you release the left mouse button, the computer calculates and
displays the new average projection. The projection axis is the viewing
direction of the user to the newly positioned image stack.
button.
Note
You can activate an average projection with the 3D Avg. button or in the
Image Tool dialog window, 3D button/ Projections and Animations icon. If
you activate the function using the button, the projection image will just
be a temporary display on the monitor. When activated through the
dialog window, the projection image will be stored as a new file in the
current experiment.


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Average Value Projection of an Image Stack with

Function
Using the button Fix. Avg. button starts an average projection. For this
of these columns of sampling points, the arithmetic average is calculated
from all intensity values and displayed in the two-dimensional projection
image as the representative of the complete column.
! Click the Fix. Avg. button. An average projection of the current image stack
is created and displayed in the Viewer window.
button.
Note
An average projection can be activated either by using the Fix. Avg.

button or in the Image Tool dialog window via 3D button, Orthogonal
Projection icon. If you activate the function using the button, the


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Transparent Projection of an Image Stack with

Function
Using the button Fix. Trans., start the transparent projection. For this
of these sampling point columns, a weighted average is calculated from
all intensity values, which is then displayed in the two-dimensional
projection image as a representative of the entire column.
The user can vary the weighting of the sampling points to calculate the
averages by setting the corresponding factors (transparent factor) in the
Viewer Options dialog window. Furthermore, you can indicate a threshold
and scale the intensity value range for the image.
! Click the Fix. Trans. button. A transparent projection of the current image
button.
Note
You can activate a transparent projection using the Fix. Trans. button.
Alternately, it can be activated using the Image Tool dialog window/ 3D
button/ Orthogonal Projection icon. If you activate the function using the
button, the projection image will just be a temporary display on the
monitor. When activated through the dialog window, the projection image
will be stored as a new file in the current experiment.


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Transparent Projection of an Image Stack with

Variable Projection Axis (optional)
Function
You can activate a transparent projection using the 3D Trans. The
projection axis is freely variable.
of these sampling point columns, a weighted average is calculated from
all intensity values, which is then displayed in the two-dimensional
projection image as a representative of the entire column.
The user can vary the weighting of the sampling points to calculate the
averages by setting the corresponding factors (transparent factor) in the
Viewer Options dialog window. Furthermore, you can indicate a threshold
and scale the intensity value range for the image.
! Click the 3D Trans. button. A transparent projection of the current image

! Upon releasing the left mouse button, the new transparent projection will
be calculated and displayed. The projection axis is the viewing direction of
the user to the newly positioned image stack.
button.
Note
A transparent projection can be activated using either the 3D Trans.

button or alternately, the Image Tool dialog window/ 3D button/
Projections and Animations icon. If you activate the function using the
button, the projection image will just be a temporary display on the
monitor. When activated through the dialog window, the projection image
will be stored as a new file in the current experiment.


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Creating SFP projections of an image stack

(optional)
Function
The SFP button starts an SFP projection, the mathematical simulation of
a fluorescence process.
projection axis are examined throughout all optical sections. In the first
step, the laser beam is simulated penetrating the sample and
diminishing. In the second step, using the determined light density, the
simulated fluorescence for each Voxel is calculated.
! Click the SFP button. A SFP projection of the current image stack is
created and displayed in the Viewer window.
! Upon releasing the left mouse button, the new SFP projection will be
calculated and displayed. The projection axis is the viewing direction of the
user to the newly positioned image stack.
button.
Note
This SFP projection can be activated using the SFP button and the
Image Tool dialog window/ 3D button/ SFP icon. If you activate the
function using the button, the projection image will just be a temporary
display on the monitor. When activated through the dialog window, the
projection image will be stored as a new file in the current experiment.


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Creating a Topographical image
Function
Use the Topography function to select specific intensity data from an
image stack, i.e. a series of xy or xz-sections, and transfer them into a
two-dimensional topographical image. This function examines the
sampling points (voxels) that are superimposed along the z-axis
throughout all optical sections. From each of these columns of sampling
points, only the intensity value that is met by the selection criterion is
displayed in the two-dimensional topographical image as the
representative of all values within the column.
You can make your selection based on either the maximum intensity or
the center of mass of the measured intensities. If you want to represent
the intensity maximums in the topographical image, only the sampling
point at which the maximum intensity was measured is selected. When
determining the center of mass, a mean value is calculated from all
superimposed sampling points (the center of mass of the area that is
limited by the curve of the measured intensity values).
The intensity maximum or center of mass is then assigned the real z

position of the corresponding sampling point and then color-coded.
Based on this assignment, a topographical image maps the real surface
structure of the specimen. Using the default settings, higher structures
appear light and deeper structures dark.
Use the Viewer Options dialog window to set the selection criterion for
the topographical image:

! Click on the Surface Calculation icon to view the Topography tab.
! see Viewer Options dialog window, Surface Calculation icon
By applying a second step, you can display the topographical image as a

three-dimensional graphical image using the 3D View function.
! see Creating the 3D View
Displaying the image data in a topographical image is especially
informative in material scientific research. For the application of
quantification functions, the topography function is indispensable.

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Viewing the Original image
Function
Click the Original button to undo a projection image or topographical
image. The Viewer window then displays the recorded raw data of the
image again.
Creating 3D Views
Creating the 3D View
Function
Use the 3D View button to display a two-dimensional data record three-
dimensionally. When viewing an image series, the data record of the
series that is currently being viewed in the Viewer window is always the
one applied. You can display a single xy-section or xz-section from the
raw data or a result image, such as a topographical image or projection
image, in the 3D view. Depending on the dimension represented or
calculated in the output image, either intensity values or height values
are mapped to the z-axis of the 3D view when the 3D view is generated.
Note
The spatial representation of intensity in a 3D view leads easily to the

assumption that the topography of the specimen is being represented.
Note however that you can only display the real surface structure of a
specimen if you have generated a topographical image first.
The 3D view can be generated in three different modes: surface,

wireframe or isolines. Set the display mode in the Viewer Options dialog
window:

! Click the Surface View icon and then click the Visualization tab.
! see Viewer Options Dialog Window, Surface View Icon

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Note
A separate 3D view is created for each detection channel. Therefore it is

not possible to generate a single 3D view from one overlay image
(Overlay button).
Rotating the 3D View
Function
Use the Rotate button to rotate a 3D view in all three spatial directions.
Note that the 3D view is rotated around a fixed point, which is located in
the center of the image.
! Hold the mouse pointer over any position in the 3D view.

! Press and hold the left mouse button.
! Move the mouse pointer in the direction in which you want to rotate the 3D
view.
Note
While the 3D view is being turned, the resolution of the image is

decreased in order to speed up processing. When the left mouse button
is released, the image is displayed with its original resolution.
As an alternative to rotating a 3D view manually, the Viewer Options

dialog window also provides the option of specifying the angles of
rotation for the three spatial axes:

! Click the 3D icon and then click the Navigation tab.
! You can then specify the angles of rotation for all three axes in the
Rotation field.
! see Viewer Options Dialog Window, 3D Icon

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Moving the 3D View
Function
Use the Move button to shift a 3D view:
! Hold the mouse pointer over any position in the 3D view.

! Press and hold the left mouse button.
! Move the mouse pointer in the direction in which you want to move the 3D
view.
Note
While the 3D view is being moved, the resolution of the image is

As an alternative to moving a 3D view manually, the Viewer Options

dialog window also provides the option of specifying the coordinate
values for the position of the image:

! Click the 3D icon and then click the Navigation tab.
! Modify the coordinate values for the x-axis and z-axis in the Translation
field.
Zooming the 3D View
Function
Use the Zoom button to magnify and shrink a 3D view steplessly while
maintaining the aspect ratio. This function only adjusts the scale of, i.e.
zooms, the generated image. It cannot be used to improve its resolution.
To zoom in Click anywhere inside the image window of the Viewer window
on the 3D and, while pressing the left mouse button, drag the mouse
view: pointer toward the lower edge.
To zoom out Click anywhere inside the image window of the Viewer window
of the 3D and, while pressing the left mouse button, drag the mouse
view: pointer toward the upper edge.

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Note
While the 3D view is being zoomed, the resolution of the image is

As an alternative to zooming a 3D view manually, the Viewer Options

dialog window also allows you to change the zoom scale by specifying
numerical values:

! Click the 3D icon.
! You can then vary the zoom factor in the Navigation tab by changing the y
value in the Translation field.
Note
The Leica Confocal Software provides three different zoom functions: 3D

zoom, electronic zoom and graphical zoom.

! see Viewer Options Dialog Window, Display Icon
Measuring and Analysis Functions

Calculating a Histogram
Function
The Histogram function measures the frequency of a measurement
variable, displays it in a curve and in addition, calculates various
statistical values. The histogram is measured from the data set selected
in the Viewer window. This data set can be a spatial series, a time series
or a wavelength series. The statistical values are calculated dependent
on the size of the third dimension; height (z) for spatial series, time (t) for
time series, wavelength (λ) for wavelength series.
! Click on the Histogram button.

! A viewer window opens that displays the measurement curve for each
detection channel and the statistical values:
Parameter Meaning Formula

# Pixel The total number of
pixels that are included in
the calculation of the
histogram. This
corresponds to the
configured scan format.

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Mean The arithmetical mean

value
Maximum Maximum value Max ( I )

Minimum Minimum value Min ( I )
Variance Variance
Average Average deviation

Deviation
Standard Standard deviation

Deviation
Mean Average image energy

Energy
Root Mean Root mean square value

Square
(RMS)
Skewness Skewness of the
distribution
Some of the parameters are specified in scientific exponential notation,

e.g. Pixel = 3.28e+005 = 327680 (corresponds to a scan format of 640 x
512).
Note
Using the maximum and minimum values of intensity, the gain values
and offset values can be optimally configured for the detectors.
Measuring a Profile Within a Region of Interest

(ROI)
Function
The Profile (z) function measures a measurement variable within a region
of interest, displays it in a curve and calculates various statistical values.
The profile is measured from the data record selected in the Viewer
window. This data set can be a spatial series, a time series or a
wavelength series. The statistical values are calculated dependent on the
size of the third dimension; height (z) for spatial series, time (t) for time
series, wavelength (λ) for wavelength series.
! Click on the Profile (z) button and draw a region of interest (ROI) in the
image. The buttons used to define a region of interest are activated
together with the Profile (z) button.
! see Defining the Region of Interest (ROI) as Ellipse
! see Defining the Region of Interest (ROI) as a Polygon

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! see Automatically Defining the Region of Interest (ROI)
! see Selecting and Shifting the Region of Interest (ROI)
! see Deleting the Region of Interest (ROI)
! A window opens automatically to display the measurement curve for every
detection channel as well as the statistical values.

# pixel (ROI) Number of pixels within the selected
region of interest
Area Area of the region of
interest
Length Height of image

stack (spatial series)
Recording time (time
series)
Bandwidth
(wavelength series)
Mean The arithmetical
Amplitude mean
Maximum Maximum value Max ( I )

Amplitude
Maximum Position of maximum Arg Max I (z), (t), (λ)
amplitude value
position
Minimum Minimum value Min ( I)
Amplitude
Minimum Position of minimum Arg Min I (z), (t), (λ)
amplitude value
position
Average Average deviation
Deviation
Standard Standard deviation

Deviation
Variance Variance
The following four measurement values appear only when the Two-
Point Measurement function is selected in the Viewer Options
dialog window.
! Select the View menu, the Experiment Overview option, and the Charts
icon.
! see Viewer Options Dialog Window, Charts Icon
! In the Measurement field you can switch two-point measurement on or off
by selecting 2 Point or Off.
l(µm); I(s); Intensity measured at measuring point 1 (z, I(z1); I(t1); I(l1)
I(nm) t, λ)
l(z); I(t); Intensity measured at measuring point 2 (z, I(z ); I(t ); I(l )
2 2 2
I(λ) t, λ)
dI, dz Difference of values between measuring |I(z1) - I(z2)|; |I(t1) -
point 1 and 2 I(t2)|, |I(l1) - I(l2)|

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d(z), (t), Height (z), recording time (t), bandwidth (λ) |z1 - z2|; |t1 - t2|, |l1 -
(λ) between the measuring points 1 and 2 l2|
This function can be used to determine the extreme values of the
intensity within an image stack. They in turn allow for the optimized
setting of the detectors. In addition, this function can be used to
determine the emission maximum of a wavelength series. By adapting
the width and the position of the screens in front of the detectors, more
emission light from the specimen can be recorded than with conventional
optical filters. In time series, this function can be used to determine the
time of the highest fluorescence activity.
Note
This quantification function is only available off-line (only available for

previously recorded data records).
Measuring a Profile Along a Line Segment
Function
The Profile function measures a measurement variable along a line
segment, displays it in a curve and calculates various statistical values.
The profile is measured from the data record selected in the Viewer
window. This data set can be a spatial series, a time series or a
wavelength series. The statistical values are calculated dependent on the
size of the third dimension; height (z) for spatial series, time (t) for time
series, wavelength (λ) for wavelength series.
! Click on the Profile button. The measurement section is displayed as a

white line in the image in the Viewer window.
You can change the length and position of the line segment by clicking the
line and, while clicking and holding the grab point with the left mouse
button, dragging it to the desired position.

Length The length of the
measured section
Mean Arithmetic mean of
Amplitude measurement variable
Maximum Maximum value of Max ( I )

Minimum Minimum value of Min ( I )

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Average Mean standard deviation

Deviation of the values from the
average value of the
measurement variable
Standard Standard deviation of
Deviation the values of the
Variance Variance of the
dialog window.
icon.

l(µm); I(s); Value measured at measuring point 1 (z, I(z1); I(t1); I(l1)
I(nm) t, λ)
l(µm); I(s); Value measured at measuring point 2 (z, I(z ); I(t ); I(l )
2 2 2
I(nm) t, λ)
dI, dz Difference of values between measuring |I(z1) - I(z2)|; |I(t1) -
point 1 and 2 I(t2)|, |I(l1) - I(l2)|
d(z), (t), (λ) Height (z), recording time (t), bandwidth |z1 - z2|; |t1 - t2|, |l1 -
(λ) between the measuring points 1 and l2|
2
Note

Measuring Surfaces and Volumes
Function
The Materials function calculates surfaces and volumes of the three-
dimensional, spatial data set.
Click on the Materials button to automatically open a window in which a
measurement curve and the size of the accumulated volume are
displayed depending on the z position. Accumulated volume means that
for each z position the value of the volume of the data set lying below it is
calculated.
A requirement for calculating the surfaces and volumes is a typographical

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image. Thus, the Materials button is only active if the Topography button
was used before to create a topographical image of the data set. Only
one detection channel can be quantified at a time.
Parameter Meaning
Scanned This value represents the horizontal projection of the surface of
Area A a xy section.
Surface This value represents the actual surface of a xy section. In
Area A' relation to the horizontal projection the actual surface is always
at least as large, in general even larger.
Ratio A'/A This value represents the relation of surface to base area. The
larger it is, the more the two-dimensional surface is embedded
in the three-dimensional space.
dialog window.
icon.
V(z<=upper This value represents the volume located below the upper
position value) position value of the measurement slider (right corner point
of the slider).
V(z<=lower This value represents the volume located below the lower
position value) position value of the measurement slider (right corner point
of the slider).
dV Volume between the position values of the slider
dz Distance between the position values of the slider
Note


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Copying Quantification Graphs to the Annotation

Sheet
Function
The Snap Quantification Data function copies the data calculated by the
quantification functions to the annotation sheet. The calculated values for
the following quantification functions, which are also displaying an
evaluation graph, can be copied to an annotation sheet:
! see Creating an Annotation Sheet
Printing Quantification Graphs
Function
The Print Quantification Data function sends the data calculated by the
quantification functions to the standard printer. The calculated values for
the following quantification functions, which are also displaying an
evaluation graph, can be printed:

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This function is also available in the context menu within the

quantification function window:
! Move the mouse pointer into the view window opened by a quantification
function.
! Click the right mouse button, and select the Print menu item.
Exporting Quantification Data
Function
Using he Export Quantification Data function, you can export the data
calculated by the quantification functions as ASCII standard data. This
enables you to read and edit data in Excel. The calculated values for the
following quantification functions, which are also displaying an evaluation
graph, can be exported:
This function is also available in the context menu within the

quantification function window:
! Move the mouse pointer into the view window opened by a quantification
function.
! Click the right mouse button, and select the Export menu item.
Defining the Region of Interest (ROI) as an Ellipse
Function
Using the Ellipse button, you can define an elliptic Region of Interest
(ROI) within the image:

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! Click the Ellipse button.

! Click the left mouse button on the image position where you would like to
place a ROI corner.
! Then, keeping the left mouse button pressed, drag the mouse pointer
diametrically to the opposite corner to define the second corner of the ROI.

! see Moving and Rotating a Region of Interest (ROI)
! see Deleting Regions of Interest (ROIs)
Other Requirements
The definition of a region of interest in the image is required for
quantification functions. Therefore, the Ellipse button is deactivated until
you activate a quantification function using one of the following buttons:
! see Measuring Roughness Within a Region of Interest (ROI)
Defining the Region of Interest (ROI) as a Polygon
Function
Using the Polygon button, you can define any polygonal Region of
Interest (ROI) within the image:
! Click the Polygon button.

place a ROI corner.
! In order to define the other polygon corners, release the mouse button,
move the cursor to the next polygon corner, and click the left mouse button
again. Repeat this step for each desired polygon corner.
! If you want to draw the polygon by hand, keep the left mouse button
pressed and draw the ROI into the image.
! Close the polygon with a double click.

Other Requirements
quantification functions. Therefore, the Polygon button is deactivated until

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Defining the Region of Interest (ROI) as a Rectangle
Function
Using the Rectangle button, you can define a rectangular Region of
! Click the Rectangle button.

place a ROI corner.
! Then, keeping the left mouse button pressed, drag the mouse pointer
diametrically to the opposite corner to define the second corner of the ROI.

Other Requirements
quantification functions. Therefore, the Rectangle is deactivated until you
activate a quantification function using one of the following buttons:

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Automatically Defining the Region of Interest (ROI)
Function
You can use the Wizard button to have the computer define any structure
in the image as a Region of Interest (ROI) automatically in the image.
Depending on which of the buttons you have clicked, an elliptical,
rectangular or polygonal region of interest is placed around the structure.
! Click on the Ellipse, Rectangle or Polygon buttons then on the Wizard

button.
! Double-click with the left mouse button on the structure in the image that
you want to mark as region of interest.
! The structure is automatically marked as an elliptical, rectangular or
polygonal region of interest.
! see Defining the Region of Interest (ROI) as Ellipse

Other Requirements
quantification functions. The Wizard button is only enabled when you
have previously enabled a quantification function using one of the
following buttons:

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Selecting and Moving the Region of Interest (ROI)
Function
Using the Select ROI button, you can select and move a Region of
! Click first the Select ROI button and then the ROI you want to move.
! Move the mouse pointer into the ROI.
! As soon as the mouse pointer changes to a crossed double arrow, press
and hold the left mouse button.
! Keeping the left mouse button pressed, move the ROI.
You can change the size of the region of interest you have drawn:
! Hold the mouse pointer over one of the ROI's edges until a double arrow
appears. Press the left mouse button to shift the shape of the ROI in one
direction.
! If you place the mouse pointer exactly over a corner of the ROI, you can
change its shape in two directions at once.

! see Deleting a Region of Interest (ROI)
Other Requirements
quantification functions. Therefore, the Select ROI is deactivated until

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Moving and Rotating the Region of Interest (ROI)
Function
Using the Rotate ROI button, you can move and rotate a Region of
! Click first the Rotate ROI button and then the region of interest you want to
rotate.
! Place the mouse pointer exactly on the corner of the selected region of
interest.
! As soon as the mouse pointer changes to a double arrow, press and hold
the left mouse button.
! Keeping the left mouse button pressed, rotate the region of interest. The
region of interest will be rotated around its center point.
You can change the size of the region of interest you have drawn:
! Hold the mouse pointer over one of the ROI's edges until a double arrow
appears. Press the left mouse button to shift the shape of the ROI in one
direction.

Other Requirements
quantification functions. Therefore, the Rotate ROI is deactivated until

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Deleting Regions of Interest (ROIs)
Function
The Clear button deletes all defined ROIs at once. To delete an individual
ROI, click the Select ROI button, then the ROI in the image, and press
the DELETE key on the keyboard.
Other Requirements
quantification functions. Therefore, the Clear is deactivated until you
activate a quantification function using one of the following buttons:
Documenting Data
Creating an Annotation Sheet
Function
Use the Annotation button to open a window in which you can prepare
recorded images for presentation purposes. If you have opened the
annotation sheet, the Snap, Line, Rectangle and Text buttons are also
enabled. Use these buttons to copy the image loaded in the Viewer
window into the annotation sheet, to highlight specific areas of the copied
image with lines and rectangles and to enter an image comment into a
text field.
! see Copying an Image

! see Drawing a Line
! see Drawing a Rectangle
! see Adding a Text Field
If you hold the mouse pointer over the annotation sheet and press the
right mouse button, a context menu containing the following commands
appears:

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Command Function
Line Use this to insert a line of default size onto the annotation sheet.
Rectangle Use this to insert a rectangle of default size onto the annotation
sheet.
Text Use this to insert a text field with a default font onto the
annotation sheet.
Zoom Use this to select from four magnifications for viewing the
annotation sheet.
Grid This displays a grid on the annotation sheet, which cannot be
printed.
The Line, Rectangle and Text commands can be activated both from the
context menu and by clicking their corresponding buttons.
Saved annotation sheets are given the *.ano file extension.
Copying an Image into the Annotation Sheet
Function
If you click the Snap button, the image loaded in the Viewer window is
copied onto the annotation sheet. If you mark the copied image and then
press the right mouse button, a context menu containing the following
commands appears:
Command Function
Original size This displays the image in its original size.
Fit to page This enlarges the image to the size of the annotation sheet.
Bring to front This brings the image to the foreground.
Send to back This sends the image to the background.
Delete This deletes the image.
You can change the size of the image by dragging one of the image grab
points. To move the image without changing its size, mark the image and
move it while pressing the left mouse button.
Note
The Snap button can be applied only if an annotation sheet has been
opened using the Annotation button first and the Viewer window has
been clicked.

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Drawing a Line on the Annotation Sheet
Function
Use the Line button to draw a line on the annotation sheet. On the
annotation sheet, click the position where you want the line to begin.
Keeping the left mouse button depressed, drag the mouse pointer across
the page to the position where you want the line to end. If you mark the
line and then press the right mouse button, a context menu containing
the following commands appears:
Command Function
Style This opens a dialog window where you can configure the style,
thickness and length of the line.
Color This opens a dialog window for selecting a color for the line.
Bring to This brings the line to the foreground.
front
Send to This sends the line to the background.
back
Delete This deletes the line.
You can change the length of the line by dragging one of its grab points.
To move the line without changing its size, click the center grab point and
move the line while pressing and holding the left mouse button.
Note
The Line button can be applied only if the annotation sheet has been
opened using the Annotation button first.
Drawing a Rectangle on the Annotation Sheet
Function
Use the Rectangle button to draw a rectangle on the annotation sheet.
On the annotation sheet, click the position where you want the corner of
the rectangle to be. Keeping the left mouse button depressed, drag the
mouse pointer across the page to define the size of the rectangle. If you
mark the rectangle and then press the right mouse button, a context
menu containing the following commands appears:
Command Function
Style This opens a dialog window where you can configure, among
other options, the style and thickness of the line.
Color This opens a dialog window for selecting a color for the
rectangle.

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Bring to This brings the rectangle to the foreground.

front
Sent to This sends the rectangle to the background.
back
Delete This deletes the rectangle.
You can change the size of the rectangle by dragging one of the image
grab points of the rectangle. To move the rectangle without changing its
size, click in the middle of the rectangle and move it while pressing and
holding the left mouse button.
Note
The Rectangle button can be applied only if the annotation sheet has
been opened using the Annotation button first.
Adding a Text Field to an Annotation Sheet
Function
Use the Text button to insert a text field onto the annotation sheet. On
the annotation sheet, click the position where you want the text field to be
placed. Keeping the left mouse button depressed, drag the mouse
pointer across the page to define the size of the text field. If you mark the
text field and then press the right mouse button, a context menu
containing the following commands appears:
Command Function
Font This opens a dialog window used to select from various fonts
and other formatting options.
Transparent This makes the background visible through the text field.
Bring to This brings the text to the foreground.
front
Send to This sends the text to the background.
back
Delete This deletes the text.
You can change the size of the text field by dragging one of the image
grab points of the text field. To move the text field without changing its
size, click in the middle of the text field and move it while pressing and
holding the left mouse button.
Note
The Text button can be applied only if the annotation sheet has been
opened using the Annotation button first.

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Printing
Function
You can trigger the print command either by clicking the Print button or
from the Printer Selection dialog window. To open this dialog window,
select the Print option in the File menu. The image loaded in the Viewer
window or an annotation sheet can be printed.
When you initiate the Print command, the image currently loaded in the
Viewer window or the currently open annotation sheet is printed using the
default printer and default page layout. You can change these default
settings in the Printer Selection dialog window to suit your needs. Use
the following buttons provided in the dialog window to do so:
Button Function
Printer setup This opens the dialog window for selecting a printer and
changing the printer settings.
Print This prints the image or the annotation sheet.
Background Use this to open a dialog window for selecting a background
color color for the page.
Center on page This centers the image on the page.
Fit to page This option changes the size of the image to fit the size of
the printable area.
Aspect ratio The height-to-width ratio remains the same when the size of
the image changes.
Use the Image field to change the height (Size Y) and width (Size X) of
the image and to define a top margin (Offset Y) and a left margin (Offset
X). The image size and margins cannot be modified if the Fit to page
option is enabled. If you have enabled Center on Page, you can change
the size but not the margins.
Check the results of your settings in the Print Preview field. Use the Page
field to specify the height and width of the printable area (which is not
identical to the paper size). The two bottom lines of the dialog window
display the currently installed printer and configured paper size.
Note
Close the Printer Selection dialog window by clicking OK to save the new
settings for the active Viewer window. This allows you to store different
printer settings and page layouts for different images. However, these
settings are not permanent and are lost when you exit the Leica Confocal
Software.

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Data Handling
Opening a File
Function
With the Leica Confocal Software, you can open various file types using
the Open button
File Type Description

Experiment A Leica-specific, binary data format. When the experiment is
(*.lei) loaded, both the image data and the experiment settings are
loaded.
Annotation A Leica-specific, binary data format. The elements on the
sheet (*.ano) annotation sheet, such as images, texts and graphic images,
are each available as individual objects.
Tiff files (*.tif) These are Leica image files in single and multi Tiff format.
External files in RGB-Tiff format can be loaded as well.

!see Creating an Annotation Sheet
Saving a File
Function
Click the Save button to save the data of the current experiment (*.lei) or
the current annotation sheet (*.ano).
If you use the Save button to save an experiment or annotation sheet for
the first time, the Save As dialog window opens.
Note
It is easy to inadvertently overwrite your original data using the Save

function. To avoid doing so, use the Save As function to save an
experiment that has already been saved once or more under a new
name.
! see Saving a File As...

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Saving a File as...
Function
Use the Save As button to save an experiment (*.lei) or annotation sheet
(*.ano) while giving it a specific name and file type.
When saving an experiment, a folder is created at the file level with the
name of the experiment. This folder then contains the description file
(*.lei) for the experiment as well as the individual image files. The format
of the description file is Leica-specific and binary. This file contains the
parameter settings and the color information (in the form of color look-up
tables) for each image belonging to the experiment.
The image files of an experiment can be saved in tif or raw format. The
standard format, tif, also contains the experiment settings and the color
information for the images. In the raw format, just the image data is
saved.
The advantage of the raw format is the smaller file size, but this is only
significant for image recordings with relatively small amounts of data. For
example, if you record an image series with a large number of individual
images but at a low scan format, you can save and open it faster in raw
format than in tif.
Storing All Data
Function
Use the Save All button to save experiments (*.lei) or annotation sheets
(*.ano) while giving them a specific name and file type.
When saving an experiment, a folder is created at the file level with the
name of the experiment. This folder then contains the description file
(*.lei) for the experiment as well as the individual image files. The format
of the description file is Leica-specific and binary. This file contains the
parameter settings and the color information (in the form of color look-up
tables) for each image belonging to the experiment.
The image files of an experiment can be saved in tif or raw format. The
standard format, tif, also contains the experiment settings and the color
information for the images. In the raw format, just the image data is
saved.

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The advantage of the raw format is the smaller file size, but this is only
significant for image recordings with relatively small amounts of data. For
example, if you record an image series with a large number of individual
images but at a low scan format, you can save and open it faster in raw
format than in tif.
Creating an Experiment
Function
Click the New Experiment button to open a new Viewer window, which
creates a new experiment. An experiment is a file that consists of one ore
more individual images or image series. This allows you to keep several
images, each recorded using different scan parameters, or edited
versions of images in one experiment. The format of the experiment file
(*.lei) is Leica-specific.
User-specific Adaptation
Saving the Viewer Window as a Template
Function
Using the Template button, you can save a user-defined design of the
Viewer window as a template. The elements of the Viewer window that
can be shown or hidden, are the buttons the color bars of the color look-
up tables and the Experiment legend.
! see Viewer Window
Clicking the Template button opens a dialog window that you can use to
enter a name for the template. This configuration of the Viewer window is
saved and will be loaded each time you open a file or create a new
experiment.
In the Tools menu, click on Options and select the Viewer Template tab
to access the user-defined templates. In the Leica Templates list box,
you can choose from the following predefined templates:
Viewer Design
Template
Viewer (Pure) The view window consists only of the image window.
Viewer (LUT) The view window consists of the image window and the color
bars of the color look-up tables.

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Viewer The view window consists of the buttons, the image window,
(Standard) the color bars of the color look-up table, and the Experiment
legend.
In the Personal Templates List box, the user-defined templates are

entered. Using the Add Active Viewer and Remove Template buttons,
you can save a new template or delete an existing one.
In the Tools menu, click on Options to indicate in the Workspace tab how
many view windows, if any, Viewer should open when the Leica Confocal
Software is started.
Controlling Functions Using the Control Panel
Function
At frequent points during an image recording, various parameters have to
be reset. For this reason, the control panel can be used to control
functions quickly and directly. You can freely allocate the radio buttons of
the control panel to a selection of functions:
! In the View menu, select the Status Bars option and then Control Panel
Status Bar.
The status bar for the control panel is displayed at the bottom of the user
interface of the Leica Confocal Software. Viewed from left to right, the
status bar consists of the icon for the control panel, the seven fields that
represent the radio buttons of the control panel, and three buttons.
! Click on one of the fields arranged in the same way as the radio buttons of
the control panel.
! A list is opened where you can select the desired function. The name of
the allocated function is shown in the field.
If you have assigned a function to all the radio buttons, you can save this
configuration as a template.
! Click on the left of the three buttons located on the right side of the status
bar.
! A dialog window is opened in which you can give the configuration a name
and save it.
! Click on the middle button to select and load a configuration.
In addition to these user-defined configurations, you can also access

other configurations predefined at the factory:
! Click on the right of the three buttons located on the right edge of the
status bar.
! This opens a dialog window listing all the configurations for the control
panel.
The configurations set as default, indicated by an L (Leica), can only be

loaded but not modified. The user-defined configurations are created in
the drop-down list under User and marked with a U (User). Click one of
the configurations and then press the right mouse button to open a
context menu, which contains the following commands:

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Command Function
Set as default The configuration is loaded as the default configuration
template when the software is started.
Remove default The setting of this configuration as default setting is
setting undone.
Load Loads the configuration.
Rename (only for Use this to change the name of the configuration.
U)
Delete (only for U) The configuration is deleted.
Optional Software packages

Materials
Image Tool dialog window, Materials button

(optional)

Filter (Linear) Icon Applying low-pass and high-pass filters to images
Filters are used to enhance the quality of an image. As a rule, this means
removing undesirable pixels from the image. The filter function available
here, a low-pass filter is used to suppress static noise in an image that
could be generated by the detector. The principle of a filter consists of

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taking the value of each pixel in an image and setting it off against the
values of the adjacent pixels. The filter kernel describes the number and
weighting of the adjacent pixels that come to bear in the calculation of the
new pixel. The kernel used here can be described using Pascal's
triangle:
Filter kernel size Pascal triangle Scaling

1 1 1/2
Kernel Size 3 1 2 1 1/4
1 3 1 3 1/8
Kernel Size 5 1 4 6 4 1 1/16
1 5 10 10 5 1 1/32
Kernel Size 7 1 6 15 20 15 6 1 1/64
1 7 21 35 35 21 7 1 1/128
Kernel Size 9 1 8 28 56 70 56 28 8 1 1/256
... ... ...
If, for example, kernel size 5 is selected, 5 pixels of the original image are
used in calculating 1 pixel in the filtered image. It is always the middle
pixel in the filter kernel that is the pixel to be calculated. In this case, the
gray values of the 5 pixels corresponding to the weighting scale 1-4-6-4-
1, are multiplied by the factors 1/16, 4/16, 6/16, 4/16, 1/16 and together
added to the final value. With respect to the calculation of the pixel C in
the following schematic representation this means that the gray values of
pixels A, B and D, E are included in the calculation of the filtered pixel C'.
C' is calculated as follows: 16x1/16 + 4x4/16 + 8x6/16 + 32x4/16 +
8x1/16 = 1 + 1 + 3 + 8 + 0,5 = 13,5
A B C D E
Gray values of pixels A-E in the original 16 4 8 32 8
image
Factors of kernel size 5 1/16 4/16 6/16 4/16 1/16
Gray value of C' in the filtered image 13,5
A calculation of this kind is performed for each pixel in the original image.
At the edge of the image, the filter mask protrudes beyond the image. For
these pixels, the value 0 is set.
! In the Kernel Size field, select a

value for the kernel size.
In the Filter Directions field, set the

dimensions of the filter. Select the
spatial axes to which the filter is to Intensity profile of an unfiltered
be applied sequentially. One- image
dimensional filters are applied, that
is, the filter is calculated only via one
axis at any given time.
Smooth filter.
The low-pass filter described above
is applied to the image.
This filter type filters the high Intensity profile of the image after
frequencies out of the image. That is, applying the Smooth filter
the very distinctive transitions from
low to high intensity values are
reduced.

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This filter can be used to remove

noise from an image.
Highpass filter.
the original image are subtracted
from the values of the low-pass- Intensity Profile of the Image After
filtered image. The result Application of the Highpass Filter
corresponds to an image that was
processed with a high-pass filter.
This filter type displays only the very

distinctive transitions from low to
high intensity values in the image.
! In the Filter Type field select the
Sharp filter.
the high-pass-filtered image are
added to the values of the original Intensity Profile of the Image After
image. Application of the Sharp Filter
This filter type filters the low
frequencies out of the image. That is,
the very distinctive transitions from
low to high intensity values are
intensified.
This filter is also used to amplify the

noise in an image.
Use the slider in the Sharp Factor

field to vary the effect of the Sharp
filter. If this factor is set to 0, the filter
is not active.
! The value specified in the Kernel Size field as Cut-off Wavelength, is the
size of the filter measured in pixels. This value is calculated on the basis of
the scan parameters (enlargement of the objective, wavelength of the
excitation light, scan format, electronic zoom factor...) used for each image.
The value specified in the Filter Directions field as Cut-off Wavelength, is
the size of the filter measured in nanometers. For example, with a value of
160 nm all wavelengths lesser than or equal to 160 nm are filtered.
! Click on the Show button to obtain a preview of the filtered images. Show
dialog window.
Or
Click on the Apply button to create the filtered images as a new data

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Measuring Roughness Along a Line Segment
Function
The Roughness Profile function measures height, distance, and angular
values of a surface along a specific line and displays these values in a
curve. The height profile is measured from the data record selected in the
Viewer window. This data record should be a spatial series.
A topographical image is the prerequisite for the generation of a height

profile. For this reason, the Roughness Profile button is active only when
a topographic image of the data record has been created in advance with
the Topography button. Only one detection channel can be quantified at
a time.
! Click on the Roughness Profile button. The measurement section is

displayed as a white line in the image in the Viewer window.
You can change the length and position of the line segment by clicking the
line and, while clicking and holding the grab point with the left mouse
button, dragging it to the desired position.

Evaluation Length of the plotted section,
length: I(P) according to DIN EN ISO 4287
Max. Peak: Height of the highest profile Max (Pp)
Pp peak (based on the average
height), according to DIN EN
ISO 4287
Min. Valley: Depth of the greatest profile Min (Pv)
Pn valley (based on the average
height), according to DIN EN
ISO 4287
Deviation: Arithmetic average of the
Pa profile ordinates within the
measured section (average
height) according to DIN EN with
ISO 4287
RMS: Pq Root mean square value of
the profile ordinates within the
measured section, according
to DIN EN ISO 4287 with

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dialog window.
icon.

h (position of the slide's Profile height at the position of the h1 ( l1 )
left measurement point) slide's left measurement point
h (position of the slide's Profile height at the position of the h2 ( l2 )
right measurement slide's right measurement point
point)
Step Height dh The profile's height difference
between the slide's left and right
measurement points
dx Distance between the slide's left and
right corners
alpha The angle of the (left measurement
point to right measurement)
connection line from horizontal
Saving the measurement values

The software can save the values on the positions of both measurement
points as well as the height difference between the measurement points.
! Select the View menu, Experiment Overview option, Surface Measure

icon.
! see Viewer Options Dialog Window, Surface Measure Icon
! To save the values, click on the Remember button in the field Multipoint
Measurement. The Clear button deletes them again.
Note


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Measuring Roughness Within a Region of Interest

(ROI)
Function
The Roughness Area function measures height, distance, and angular
values of a surface within a region of interest. The profile is measured
from the data record selected in the Viewer window. This data record
should be a spatial series.
A topographical image is the prerequisite for the generation of a height

profile. For this reason, the Roughness Area button is active only when a
topographic image of the data record has been created in advance with
the Topography button.
! Click on the button Roughness Area and draw a region of interest (ROI)
into the image. The buttons with which you define a ROI are activated
together with the Roughness Area button.
! see Defining the Region of Interest (ROI) as an Ellipse
! see Automatically Defining the Region of Interest (ROI)
! A window opens automatically to display the statistical values.

# Pixel Number of pixels within the selected
region of interest
Area Area
Avg. Average height of the profile ordinates

Height
Pa Arithmetic average of the profile

ordinates within the measured section
(average height) according to DIN EN
ISO 4287
with
RMS (Pq) Root mean square value of the profile
ordinates within the measured section,
according to DIN EN ISO 4287
with
Min Valley Minimal height of the profile ordinates Min (Pv)

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(Pn) (based on the average height)

Max Peak Maximal height of the profile ordinates Max (Pp)
(Pp) (based on the average height)
Note


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Glossary
Glossary
Aberration, An optical image distortion conditional on the varying refraction of light rays of
chromatic different wavelengths on a lens. Thus light rays of shorter wavelengths have
longer focal distances than light rays of longer wavelengths.
Aberration, An optical image distortion conditional on the varying distance of paraxial light
spherical rays of the same wavelength from the optic axis. Light rays that travel through
outer lens zones have shorter focal distances than rays that travel through the
lens center (optic axis).
Achromatic Describes a correction class for objectives. The chromatic aberration for two
wavelengths is corrected for objectives of this type. Usually an objective of this
type is corrected to a wavelength below 500nm and above 600nm.
Furthermore, the sine condition for one wavelength is met. The image
curvature aberration is not corrected.
Airy disc The Airy disc refers to the inner, light circle (surrounded by alternating dark
and light diffraction rings) of the diffraction pattern of a point light source. The
diffraction discs of two adjacent object points overlap some or completely,
thus limiting the spatial resolution capacity.
Aliasing An image distortion caused by a sampling frequency that is too low in relation
to the signal frequency.
AOTF The acousto-optical tunable filter is an optic transparent crystal that can be
Acousto-Optical used to infinitely vary the intensity and wavelength of radiated light. The
Tunable Filter crystal generates an internal ultrasonic wave field, the wavelength of which
can be configured to any value. Radiated light is diffracted vertically to the
ultrasonic wave field like through a grid.
Aperture, Aperture is the sine of the opening angle under which light enters into the front
numerical lens of a microscope objective; its symbol is NA. The aperture influences both
the light intensity and the resolution capacity of an objective optical system.
Since various media can be present between specimen and objective lens
(such as the embedding medium for the specimen), the numerical aperture
(NA = n * sin a) is usually applied as the unit of measurement for the light
intensity and the resolution capacity.
Apochromatic Describes a correction class for objectives. The chromatic aberration for three
wavelengths is corrected for objectives of this type (usually 450nm, 550nm
and 650nm) and the sine condition for at least two colors is met. The image
curvature aberration is not corrected.
Working The distance from the front lens of an objective to the focal point. For a
distance variable working distance, the gap between the front lens of the objective and
the cover slip or uncovered sample is specified. Usually objectives with large
working distances have low numerical apertures, while high-aperture
objectives have small working distances. If a high-aperture objective with a
large working distance is desired, the diameter of the objective lens has to be
made correspondingly large. These, however, are usually low-correction optic
systems, because maintaining extreme process accuracy through a large lens
diameter can only be achieved with great effort.
Image curvature The curved surface to which a microscopic image is to be clearly and distinctly
aberration mapped is described as image curvature aberration. It is conditional on the
convex shape of the lens and makes itself apparent as an error due to the
short focal distances of microscope objectives. Here the object image is not in
focus both in the center and at the periphery at the same time. Objectives that
are corrected for image curvature aberration are called plane objectives (plane
= flat image field).
Bleaching, The destruction of fluorochromes, so-called fluorochromes, by intense lighting.
optical In fluorescence microscopy, fluorochromes are excited with laser light to a
high state of energy, the Singlet state. When the excited molecules return to
their normal energy state, a fluorescent signal is emitted. If the intensity of the

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Glossary
excitation is too high however, the color molecules can change via
intercrossing from a Singlet state to a triplet state. Due to the significantly
longer life of triplet states (phosphorescence), these excited molecules can
react with triplet-oxide and be lost for further fluorescence excitation.
Refraction index The factor by which the light velocity in an optical medium is less than in a
vacuum.
Dichroic Dichroic filters are interference filters at an angle of incidence of light of 45°.
The transmissivity or reflectivity of dichroites depends on a specific
wavelength of light. For an RSP 510 filter (reflection short pass) ), for
example, the excitation light below 510 nm is reflected and the excitation light
above this value is transmitted. The transmission values are generally
between 80% and 90% and the reflection values between 90% and 95%.
Double dichroite Double dichroic filters are interference filters at an angle of incidence of light of
45°. The transmissivity or reflectivity of double dichroites depends on two
specific wavelengths of light. For a doubledichroite DD 488/568, for example,
the excitation light at 488 nm and 568 nm is reflected and the excitation light
above these values is transmitted. The transmission values are generally at
80% and the reflection values are between 90% and 95%.
Filter, digital, A digital filter consists of a computing rule used to modify image data. Filters
phase-true are always applied to remove disturbing image components. A phase-true
filter ensures that quantifiable image values do not change through filtering
and remain a requirement for standardized measuring methods (e.g.,
characterization of surfaces in accordance with ISO).
Fluorescence A light-optical contrast process for displaying fluorescent structures. Auto-
microscopy fluorescent samples have a so-called primary fluorescence. They do not need
to be enriched with additional, fluorescent substances. Secondary fluorescent
substances, on the other hand, have to be treated with appropriate dyes or
stains called fluorochromes. Specific dyeing methods additionally allow the
precise localization of the stained structure elements of an object.
Fluorescence microscopy allows both potential morphological examinations
and the ability to carry out dynamic examinations on a molecular level.
Fluorite Describes a correction class for objectives. Fluorite objectives are semi-
objectives apochromatic, meaning their degree of correction lies between the achromatic
and apochromatic.
Frame A frame corresponds to the acquisition of a single optical section. For
example, if a single optical section is acquired 4 times (to average the data
and to eliminate noise), then 4 frames are created for this optical section.
Immersion A microscopic objective, developed with the requirements for applying
objective immersion media. The use of incorrect or no immersion medium with an
immersion objective can lead to resolution loss and impairment of the
correction.
Confocality While the optical design of conventional microscopes allows the uniform
detection of focused and unfocussed image components, the confocal
principle suppresses the structures found outside of the focal plane of the
microscope objective. Screens are implemented in optically conjugated
locations to achieve this. They function as point light source (excitation
screen) and point detector (detection screen). The diameter of the detection
screen, along with the wavelength and numerical aperture of the objective
being used, determines the axial extension of an optical section (optical
resolution).
Reflection short Reflection short pass filters are interference filters that transmit short-wave
pass filter light while reflecting long-wave light. An optical reflection short pass filter is
characterized by the reading of the wavelength edge at which the filter
changes from transmission to reflection (50% threshold).
Reflection long Reflection long pass filters are interference filters that reflect short-wave light
pass filter but are transparent for long-wave light. An optical reflection long pass filter is
characterized by the reading of the wavelength edge at which the filter
changes from reflection to transmission (50% threshold).
Empty A magnification without additional gain of information. Empty magnification is

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Glossary
magnification used as soon as distances are displayed that are smaller than the optical
resolution. Magnifications with a larger scale than that of the empty
magnification do not provide any additional information about the object but,
instead, only diminish the focus and the contrast.
Neutral filter Neutral filters are semi-reflective glass plates. They are used to distribute the
light path independent of wavelength. The incident light is partially reflected
and partially transmitted. Neutral filters are usually placed at a 45° angle in the
path of the beam. The ratings of a neutral filter are based on its reflectivity-to-
transmissivity ratio. A neutral filter RT 30/70, for example, reflects 30% of the
excitation light and transmits 70%.
Phase The principle of phase visualization as used by Leica is an optimized
visualization alternative method to ratiometric displaying. The main area of application lies
in measuring ion concentrations in physiology. In contrast with ratiometric
procedures, phase visualization obtains more information on the specimen. In
addition, this method allows for adapting the display of physiological data to
the dynamics of the human eye. Detailed information on the principle of phase
visualization can be obtained directly from Leica Microsystems Heidelberg
GmbH.
Pixel An acronym based on the words, picture and element. A pixel represents the
smallest, indivisible image element in a two-dimensional system. In this
documentation, both the sampling points of the specimen as well as the image
points are referred to as pixels.
Plane objectives Describes a correction class for objectives. The image curvature aberration is
corrected for objectives of this type. Correcting this error requires lenses with
stronger concave surfaces and thicker middles. Three types of plane
objectives, plane achromate, plane apochromate and plane fluorite, are based
on the type of additional correction for chromatic aberration.
ROI Abbreviation for "Region of Interest". ROI encloses an area for which a
measurement analysis is to be performed. On top of that, an ROI can also
designate the area of a specimen to be scanned (ROI scan).
Signal-to-noise The ratio of signals detected in the specimen to the unwanted signals that are
ratio caused randomly by various optic and electronic components, which are also
recorded by the detector.
Stokes shift The Stokes shift is a central term in fluorescence microscopy. If fluorescent
molecules are excited with light of a specific wavelength, they radiate light of
another, larger wavelength. This difference between excitation light and
fluorescent light is referred to as Stokes shift. Without Stokes shift, separating
the high-intensity excitation light from the low-intensity fluorescence signals in
a fluorescence microscope would not be possible.
Triple dichroic Triple dichroic filters are interference filters at an angle of incidence of light of
45°. The transmissivity or reflectivity of triple dichroites depends on three
specific wavelengths of light. For a tripledichroite TD 488/568/647, for
example, the excitation light at 488 nm, 568 nm and 633 nm is reflected and
the excitation light above these values is transmitted. The transmission values
are generally at 80% and the reflection values are between 90% and 95%.
Dry objective A microscopic objective used without immersion media. Between the objective
lens and the specimen is air.
Voxel An acronym based on the words, volume and pixel. A Voxel represents the
smallest, indivisible volume element in a three-dimensional system. In this
documentation, both the volume elements of the specimen as well as the 3D
image points are referred to as voxels.

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Appendix
Appendix
Adaptation Function For Correcting Trend Curves
1. Selecting the step width, that is, the distance of the support positions
2. Selecting the character of the interpolation function to be adapted
3. Calculating the interpolation function f(x) = a + bx + cy
4. Adding the intensity differences to the leveling of the trend curve
before leveling
after leveling

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Appendix
Example of moving an image in the result image
Example of merging images with different numbers of channels
a.) First output image (setting: Start=2)

b.) Second output image (setting: Start=1)
c.) Merged image

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Appendix
How Boolean Operations Work
AND Write a 1 if
there is a 1 in
pixel 1 AND in
pixel 2;
otherwise write
0 AND represents the
intersection of two images
a.) Binary
representation of
pixel 1
b.) Binary
representation of
pixel 2
c.) Binary
representation of
result pixel
OR Write a 1 if
there is a 1 in
pixel 1 OR in
pixel 2;
otherwise write
0
OR represents the union of
sets of two images
a.) Binary
representation of
pixel 1
b.) Binary
representation of
pixel 2
c.) Binary
representation of
result pixel
XOR Write a 1 if the
values in pixel 1
and in pixel 2
differ; otherwise
write 0
XOR represents the union of
sets without the intersection
of two images.
a.) Binary
representation of
pixel 1
b.) Binary
representation of
pixel 2
c.) Binary
representation of
result pixel

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Appendix
LCS File Formats
Formats of User-specific and Device-dependent

Data
The LCS software provides the following file formats for user-specific
data:
Directory: Leica Directory: User

Instrument data (Instrument *.IPS *.IPS
Parameter Settings)
Macros Company.mac User.mac
Templates for operator console *.pbo *.pbo
User profiles CompanyProfile.pro PersonalProfile.pro
LastExitProfile.pro
LastRecent.lst
Templates *.prt *.prt
Calibration data Machine.lhw
Color Look-Up Tables *.lut
User-specific profile as it was *.lst
defined the last time in use
In detail, the different file formats contain:
*.IPS
Instrument parameters are saved in this format. This includes hardware-
based settings such as intensity of the individual laser lines, number of
channels used simultaneously, type of main beam splitter used, position
and bandwidth of individual spectrophotometer diaphragms and
sensitivity of detectors. You can use factory-defined standard sets of
instrument parameters that cannot be changed but that allow for a very
quick configuration of the hardware. In addition, every user can save
instrument parameter sets in a separate user directory. This allows you
to save the instrument parameters typical for your planned use so that
they can be reproduced and made available for other experiments.
*.mac
Macros are saved in this format. Macros contain VBA (Visual Basic for
Application) program code. Leica provides the individual program objects
for controlling the confocal system in an object model. Additional
information about the development of macros for controlling the confocal
system can be found in the chapter on "The LCS Macro Language".
The software includes factory-predefined macros that can be used
without modifications or modified according to your particular needs. The
self-designed or modified macros are stored in the respective user-
defined directory.
*.pbo
Templates for the assignment of the control panel are saved in this
format. These templates define which scan parameter is controlled by
which rotary knob. This format also includes factory presets that have
proven useful for most standard applications. Self-defined templates are
saved in the user-specific directory.
*.pro
Specific profiles of the user interface are saved in this format. This
includes information on the type of key and its location in a specific
toolbar. In addition, new toolbars can be defined or existing defined
toolbars can be hidden or displayed. The position of the toolbars–

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Appendix
whether embedded or freely moveable–is also stored in a profile. This

allows every user to save an individual user interface. For example,
system administrators can use this tool to create a separate user
interface for each knowledge level of the user (beginner, advanced,
expert).
*.lst
Besides the individual user-specific profiles, the user directory also
contains the profile of the last session as well as a list of the last four
data records applied by the user.
*.prt
Templates for the "Viewer" window are saved in this format. This allows
for specifically saving the appearance and type of displayed information.
An explanation of the individual configuration options can be found in the
chapter on "Viewer window".
*.lhw
Calibration data and hardware settings of the confocal system are saved
in this format. This file may be edited only by service personnel certified
by Leica Microsystems Heidelberg.
Warning
Changes to this file may result in serious, possibly irreparable damages

to the confocal system. Damages demonstrably resulting from changes
to this file by the user will result in a loss of warranty for the complete
system.
*.lut
Color look-up tables are saved in this format. They are used to assign the
intensity values measured by the detectors to each color. This allows to
bring out not only the true color display, but also certain intensity areas.
More details can be found in the chapter on "Selecting Color Look-Up
Tables."
Fixed Leica-specific File Formats
The following file formats can be opened and viewed in the Leica
Confocal Software:
Raw data (*.raw)
The RAW format saves data as a linear two-dimensional array in the
INTEL format. The column index of the array corresponds to the fastest
scan dimension (in general the x-axis), while the line index corresponds
to the slower scan dimension (in general the y-axis). In an 8-bit image,
each measured variable is saved as a single byte. A 12-bit image uses 2
bytes, whereby the first byte contains the higher-valued bits in
accordance with the INTEL format (little endian). Each optical section is
saved for each channel in a separate file with the file extension .raw
using the following naming convention: name of experiment_name of
data record_channel number_z-dimension_number of optical
sections.raw
Experiments (*.lei)
The format of this type of file is Leica-specific and binary. This format is
provided for the data of entire experiments.
A file description is saved in this file format, referencing a series of image
files in TIF format or RAW format.
Tiff files (*.tif)
These are Leica image files in single and multi TIFF format. Both image
files in the previously used TCS formats and external files in RGB TIF

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Appendix
format can be read.

Annotation (*.ano)
The format of this type of file is also Leica-specific and binary. It is used
for saving annotation sheets. The elements on the annotation sheet,
such as images, texts and graphic images, are each available as
individual objects.
Note
Information on whether the stored measured variable is an intensity value

or a height value is saved in the corresponding LEI file.
Specification of the "Lei" file format version: beta 2.000
When a file is saved in the LCS software, a subdirectory is first created

under the current directory.
The following files are saved in this subdirectory:
1. A description file– its structure is described below. This file is used for
interpreting the data of an experiment through the LCS software.
2. The individual image files – the following convention is used in
assigning the name of the image files:
NAME_channel number_dimensionIndex. Thus, Chloroplast_C1_Z1
means that "Chloroplast" is the name of the data record assigned by the
user, that the image corresponds to the first optical section of a three-
dimensional data stack (Z1), and that the data was acquired in the first
detection channel (C1).
Structure of description file
The description file can be read only by the LCS software.

It consists of a collection of various tables and contains the following
structure:
Header table
Byte Meaning Possible values
0 ... 3 Byte arrangement 0x49494949 Intel LSB
0x4D4D4D4D Motorola MSB
4 ... 7 Identification for Leica 0x016033F0
"Lei" format
8 ... Version number current: =x20000000
11
12 ... Address of directory table any possible address within the complete
15 A length of the file
Directory table A
Address Meaning Entry data
type
Address of Number of entries in this table DWORD
A=A
A+4 Index of the first entry (represents a logical memory DWORD
block, e.g., acquisition parameter ("hardware
settings") or image parameter ("dimensions"))
A+8 Address A1 of the 1st entry DWORD
A+12 Index of the first entry (represents a logical memory DWORD
block, e.g., acquisition parameter ("hardware
settings") or image parameter ("dimensions"))

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
A+14 Address A2 of the 2nd entry DWORD

… ... ...
The length of the directory table depends upon the number of images
and image data to be saved in an experiment (acquisition parameters,
number of channels, number of optical sections, etc.).
Each entry in the directory table corresponds to a logical memory block.
Block table Xn
Address Meaning Data type
A1 or A2 or Check digit DWORD
A3.....Ai
Ai+ 4 bytes Description of the contents of the DWORD
block table (currently not used)
Ai+ 8 bytes Version of entry DWORD
Ai+ 12 bytes Size of entry DWORD
Ai+ 16 bytes Begin of entry The data type depends
upon the entry type.
The following logical memory blocks are available:

const DWORD ID_SERIES = 10;
const DWORD ID_IMAGES = 15;
const DWORD ID_DIMDESCR = 20;
const DWORD ID_FILTERSET = 30;
const DWORD ID_TIMEINFO = 40;
const DWORD ID_SCANNERSET = 50;
const DWORD ID_EXPERIMENT = 60;
const DWORD ID_LUTDESC = 70;
A memory space of 32 bits is defined for the DWORD data type.
ID_SERIES memory block

This data block contains information about the size of the complete data
series combined in an experiment. It always occurs only once per
experiment and is structured as follows:
Size Data Symbol Description

[byte] type
4 int Internal version number
4 int nSE Number of image series
4 int nIm Length of a file name in wchar
4 int nExt Length of the file extension of the image file
in bytes
nExt wchar File extension of image file
ID_IMAGES memory block

This block contains all file names of all image files that are part of a
series. The data block is structured as follows:
4 int nFiles Number of single images of

the series
4 int Width of a single image
4 int Length of a single image
4 int Bits / data points (image
resolution)
4 int Data points / pixels (display
resolution)
For the next n

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
image files
nIm * wchar Name of the next image
2
ID_DIMDESCR memory block

This block provides a description of an image in an n-dimensional space.

[byte] type
4 int Internal version number of the
VOXEL description
4 int Voxel type, e.g., RGB or GRAY
(see the following VOXEL
TYPES table)
4 DWORD Byte size of a pixel
Typical: (1 or 2) or (3 or 6 for
RGB)
4 DWORD Resolution of scan data (8, 12 or
16 bit)
4 int nTC Length of the following string in
wchar (wchar = wide character);
character format that uses a 2-
byte coding of characters unlike
the ASCII format (1 byte).
nTC * wchar Maximum value of measured
2 variable (intensity value or
length value of a distance) for a
voxel.
wchar
nTC * wchar Minimum value of measured
2 variable (intensity value or
length value of a distance) for a
voxel.
wchar
nTC * wchar Designation of measured
2 variable ("I" for intensity, "z" for
length value)

4 int nDims Dimension of the image, e.g.,
x_y_ch_z = 4
For the next
n dimensions
4 DWORD Identification number (ID) of the
dimension (see the following list
"Identification numbers (ID) for
image dimensions")
4 DWORD Size of dimension (e.g., 512
pixel)
4 DWORD Distance between sub-
dimensions (e.g., byte distance
between the image series of two
consecutive channel numbers)
wchar
nTC * wchar Physical length with unit of
2 length, e.g., 10 µm

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix

wchar
nTC * wchar Physical start position with unit
2 of length, e.g., 10 µm
bytes
nTC wchar Name of the image series
bytes
nTC wchar Description of the image series
Identification numbers (ID) for image dimensions

The following identification numbers are possible:
ID (decimal) Meaning
0 undefined
120 x
121 y
122 z
116 t for time dimension
6815843 channel number
6357100 wavelength Range
7602290 rotation
7798904 x-wide for motorized xy-stage
7798905 y-wide for motorized xy-stage
7798906 z-wide for z-stage
4259957 user1 (not specified)
6357095 gray-scale, e.g., of histogram

6422631 gray-scale1, e.g., of histogram
7864398 logical x (not physical, but logical position value)

7929934 logical y (not physical, but logical position value)
7995470 logical z (not physical, but logical position value)
7602254 logical t (not physical, but logical position value)
7077966 logical wavelength value (not physical, but logical value)
7471182 logical rotation value (not physical, but logical value)
5767246 logical x-wide value (not physical, but logical value)
5832782 logical y-wide value (not physical, but logical value)
5898318 logical z-wide value (not physical, but logical value)
ID_FILTERSET memory block

This memory block describes the hardware settings such as pinhole
diameter or selected filters. The memory block starts with a SAFEARRAY
header that describes the number and size of the entries. A complete
description of the SAFEARRAY structure can be found in Appendix 1.
Presently, only three entries of the SAFEARRAY structure are used:
1. sa.cDims should = 1. Presently, only one-dimensional

data fields (arrays) are used.
2. sa.cbElements Size of elements in byte
3. sa. rgsabound[0].cElements indicates the number of elements
contained in the structure

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
Size Data type Symbol Description

[byte]
24 SAFEARRAY sa Microsoft
type, see
Appendix 1
for all elements that contain the
structure =
sa.rgsabound[0].cElements
128 wchar Identifier
for the
contents of
the entry
64 wchar Name of a
short
description,
e.g., a
TIFF
description
tag
64 wchar String
(contains
the
contents of
the string)
16 VARIANT Contains
the data
type and
the data
value
(except for
the "string"
data type
which is
described
in the field
above)
4 DWORD Separate
memory
space for
data values
4 DWORD Not in use
4 DWORD Identifier
(ID) of the
memory
block (for
internal
use only)
4 DWORD Test value
ID_TIMEINFO memory block

This memory block contains the time taggers for image series that were
acquired with a time-dependent scan mode.

[byte] type
4 int nDims Indicates the number of

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
dimensions that the

description of the time
tagger includes
4 int Indicates which dimension
was provided with a time
tagger
For the
next n
dimensions
4 DWORD Identifier (ID) of the
dimension (see the
description of the
D_DIMDESCR memory
block)
4 DWORD Size of the dimension
(number of elements, e.g.,
512 pixel)
4 DWORD Distance between the
individual dimension entries
4 int nTS Number of time taggers
For the
next ntime
taggers =
nTS
64 wchar Time tagger as string
("string" data type)
4 int nTM Number of time tags per
time tagger
For the
next m
time tags =
nTM
4 int nC Number of
dimensions
of the time
tagger
description
loop
nC
4 int Coordinate
in this
dimension
64 wchar Time tag
as string
(wchar
data type)
Example:
Image with the following dimensions and dimension sizes:

x y ch z
512 512 3 10
For example, the description of a time tag is 512_512_3_10. This

description matches the description of the image. The time tagger
description 1024_1024_3_10 for the above sample image means that the
original image featured an xy-dimension of 1024*1024 and was reduced
by the software (downsampling). In such cases, the time tag must be re-
calculated accordingly.

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
A time tag can be defined at any time during an n-dimensional data

acquisition process (scan). The coordinate of the time tag 0_0_2_7
indicates that the time tag was set at the start of a data record (0_0_c_#)
on the second channel (0_0_2_#) for the 7th optical section (0_0_2_7).
ID_SCANNERSET memory block

This memory block contains device parameters that are set at the time of
the image acquisition.
Further details about the SAFEARRAY structure can be found in the
description of the ID_FILTERSET memory block.
Size Data type Symbol Description

[byte]
24 SAFEARRAY sa Microsoft type, see Appendix
for all elements that contain the structure =
sa.rgsabound[0].cElements
128 wchar Identifier for the contents of this entry
64 wchar Name of a shorthand notation, e.g., a
TIFF description tag
64 wchar String (contains the contents of the
string)
20 VARIANT Contains the data type and the data
value (except for the "string" data
type which is described in the field
above)
4 DWORD Separate memory space for data
values
4 DWORD Not in use
4 DWORD Identifier (ID) of the memory block
(for internal use only)
4 DWORD Test value
ID_EXPERIMENT memory block

Description of the used save format (e.g., “*.lei file with PC-Tiff images”)

[byte] type
4 int Number of images in the data collection of
the experiment
4 int nTC Length of the following string in wchar
nTC * 2 wchar Short description of the format
nTC * 2 wchar File extension of the main file
nTC * 2 wchar Identifier of the single image format (e.g.,
PC-TIFF)
nTC * 2 wchar File extension for the single image format
(e.g., tif / raw)
ID_LUTDESC memory block

Description of the color look-up tables (LUT's)

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix

[byte] type
4 int nLU Number of channels
4 DWORD Identifier (ID) of the
dimension whose display is
being colored.
For the next
channels until =
nLU
1 bool bool IsInverted
4 int nTC Length of the following
string in bytes
nTC wchar Description of the color
look-up table (LUT)
string in bytes
nTC wchar File name of the color look-
up table (if available)
string in bytes
nTC wchar Name of the color look-up
table
4 int Field for internal use
4 int Dimension of the color look-
up table
Appendix 1
SAFEARRAY Data Type

typedef struct FARSTRUCT tagSAFEARRAY {
unsigned short cDims; // Count of
dimensions in this array
unsigned short fFeatures; // DON’T CARE
unsigned long cbElements; // Size of an element
of the array.
// Does not include
size of
// pointed-to data.
unsigned long cLocks; // DON’T CARE
void HUGEP* pvData; // Pointer to the
data.
SAFEARRAYBOUND rgsabound[1]; // One bound for each
dimension.
} SAFEARRAY;
Important: The Leica-specific file format "LEI" uses only one-

dimensional data fields (arrays).
SAFEARRAYBOUND Structure
Represents the bounds of one dimension of the array. The lower bound

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
of the dimension is represented by lLbound, and cElements

represents the number of elements in the dimension. The structure is
defined as follows:
typedef struct tagSAFEARRAYBOUND {
unsigned long cElements; // num of
elements
long lLbound; // DON’T CARE
} SAFEARRAYBOUND;
VARIANT and VARIANTARG

typedef struct FARSTRUCT tagVARIANT VARIANT;
typedef struct tagVARIANT {

VARTYPE vt;
unsigned short wReserved1;
union {
unsigned char bVal; // VT_UI1.
short iVal; // VT_I2
.
long lVal; // VT_I4
.
float fltVal; // VT_R4
.
double dblVal; // VT_R8
.
VARIANT_BOOL boolVal; //
VT_BOOL.
SCODE scode //
VT_ERROR.
CY cyVal; // VT_CY
.
DATE date; //
VT_DATE.
BSTR bstrVal; //
VT_BSTR.
IUnknown FAR* punkVal; //
VT_UNKNOWN.
IDispatch FAR* pdispVal; //
VT_DISPATCH.
SAFEARRAY FAR* parray; //
VT_ARRAY|*.
unsigned char FAR* pbVal; //
VT_BYREF|VT_UI1.
short FAR* piVal; //
VT_BYREF|VT_I2.
long FAR* plVal; //
VT_BYREF|VT_I4.
float FAR* pfltVal; //
VT_BYREF|VT_R4.
double FAR* pdblVal; //
VT_BYREF|VT_R8.
VARIANT_BOOL FAR* pboolVal; //
VT_BYREF|VT_BOOL.
SCODE FAR* pscode; //
VT_BYREF|VT_ERROR.

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
CY FAR* pcyVal; //
VT_BYREF|VT_CY.
DATE FAR* pdate; //
VT_BYREF|VT_DATE.
BSTR FAR* pbstrVal; //
VT_BYREF|VT_BSTR.
IUnknown FAR* FAR* ppunkVal; //
VT_BYREF|VT_UNKNOWN.
IDispatch FAR* FAR* ppdispVal; //
VT_BYREF|VT_DISPATCH.
SAFEARRAY FAR* FAR* pparray; //
VT_ARRAY|*.
VARIANT FAR* pvarVal; //
VT_BYREF|VT_VARIANT.
void FAR* byref; // Generic
ByRef.
};
};
Important: Files with the "LEI" format never use pointers as parameters.
VARTYPE
typedef unsigned short VARTYPE;
enum VARENUM{
VT_EMPTY = 0, // Not specified.
VT_NULL = 1, // Null.
VT_I2 = 2, // 2-byte signed
int.
VT_I4 = 3, // 4-byte signed
int.
VT_R4 = 4, // 4-byte real.
VT_R8 = 5, // 8-byte real.
VT_CY = 6, // Currency.
VT_DATE = 7, // Date.
VT_BSTR = 8, // Binary string.
VT_DISPATCH = 9, // IDispatch
VT_ERROR = 10, // Scodes.
VT_BOOL = 11, // Boolean; True=-1,
False=0.
VT_VARIANT = 12, // VARIANT FAR*.
VT_UNKNOWN = 13, // IUnknown FAR*.
VT_UI1 = 17, // Unsigned char.
// Other constants that are not valid in VARIANTs

omitted here.
};
VT_RESERVED = (int) 0x8000
// By reference, a pointer to the data is passed.
VT_BYREF = (int) 0x4000
VT_ARRAY = (int) 0x2000 // A safe array of
the data is passed.
The vt value determines the interpretation of the data of the experiment

as follows:
Value Description

Art.No.: 15-9330-033 / Vers.: 01082001
Appendix
VT_EMPTY No value was entered

VT_UI1 An unnamed 1-byte character was saved in bVal.
VT_I2 A 2-byte integer value was saved in iVal.
VT_I4 A 4-byte integer value was saved in lVal.
VT_R4 An IEEE 4-byte real value was saved in fltVal.
VT_R8 An IEEE 8-byte real value was saved in dblVal.
A fixed point value is indicated. It consists of 15 digits before the
VT_CY
point and 4 digits after the point. The value was saved in cyVal.
Exceptions for files in LEI format:
VT_BSTR
A string was saved in the variable TCHAR
A floating zero value was indicated. Here, the floating zero is not a
VT_NULL NULL pointer. This value is required for a 3-state logic, for
example, in the SQL database query language.
An SCODE error was indicated. The error type is specified
VT_ERROR
scodee.
A Boolean value (true / false) was indicated.
VT_BOOL The value 0xFFFF (all bits=1) means "true," the value 0 (all
bits=0) means "false."
A value corresponding to a date was indicated.
A date is saved as a number in double-precision format. All data is
VT_DATE
saved as differential dates in reference to January 1, 1900.
For example, the entry "25" corresponds to 01-25-1900.

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Index
*.ano ................................................... 127, 128 Apochromatic ..............................................138
*.lei.............................................. 127, 128, 129 Apply .............................................................45
*.tif....................................................... 127, 128 Apply Button ..................................................45
12 Bit....................................................... 47, 77 Applying the Parameter Setting of an

Experiment................................................45
2-Point .......................................................... 53
Area .............................................................136
3D ................................................................. 51
Arithmetic ......................................................77
3D Display .................................................. 106
Arithmetic Average ..................... 109, 134, 136
3D image data record ................................... 41
Arithmetic With Constant...............................77
3D View Button........................................... 106
ASCII Data ..................................................116
8 Bit......................................................... 47, 77
Automatically Defining the Region of Interest
Aberration ................................................... 138 ................................................................119
Absorption Coefficient .......................... 92, 104 Average ...................................................46, 77
Achromate .................................................. 138 Average deviation................................109, 112
Achromatic.................................................. 138 Average image energy ................................109
Acousto-Optical Tunable Filter ................... 138 Average Projection56, 64, 92, 95, 96, 100, 101
Adding a Text Field..................................... 125 Averaging Method .........................................46
Adding a Text Field to an Annotation Sheet125 Basic Settings................................................51
Adding Images.............................................. 77 Beam Path Setting Dialog Window .. 22, 37, 39
Adjusting phase ............................................ 44 Begin Button..................................................37
Airy Disc ............................................... 29, 138 bidirectional ...................................................43
Aliasing ................................................. 30, 138 bi-directional ..................................................44
AND ............................................................ 143 Bidirectional scan ..........................................43
Angle of Rotation .......................................... 44 Bilder addieren ..............................................77
Annotation .......................... 122, 123, 124, 125 Bilder dividieren.............................................77
Annotation Button ....................................... 122 Bilder multiplizieren .......................................77
Annotation Sheet ........ 115, 122, 123, 124, 125 Bilder subtrahieren ........................................77
AOTF .......................................................... 138 Bleaching...............................................35, 138
Apertur ........................................................ 138 Boolean Operations ....................................143
Aperture ...................................................... 138 Burst mode ....................................................46
Apochromate .............................................. 138 Calculating...................................................109

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Center of mass ........................................... 105 Displaying an Image From a Series ..............90
Center of mass of intensities ...................... 105 Displaying the Previous Image in a Series ...89
Channel ........................................................ 83 Dividing Images.............................................77
Clear ........................................................... 122 Documentation Conventions .........................21
Color Mixing.................................................. 87 Double dichroic............................................138
Configuring a Time Series ............................ 33 Double dichroic filters ....................................22
Confocality.................................................. 138 Drawing a Line ............................................124
Continuous Scan .......................................... 35 Drawing a Line on the Annotation Sheet ....124
Contrast Transfer Function........................... 77 Drawing a Rectangle ...................................124
Control Panel.............................................. 130 Drawing a Rectangle on the Annotation Sheet

................................................................124
Conversion ................................................... 77
Dry Objective.........................................24, 138
Coordinate Axes ........................................... 51
Dynamic average ..........................................46
Copying ...................................................... 123
Edit Legend Dialog Window ..........................48
Copying an Image into the Annotation Sheet
............................................................... 123 Electronic Zoom ............................................26
Copying Quantification Graphs .................. 115 Ellipse..........................................................116
Correcting Trend Curves ...................... 71, 141 Empty Magnification ....................................138
Creating a Topographical Image .......... 61, 105 End Point.......................................................36
Creating an Experiment.............. 127, 128, 129 End Point for a Spatial Series .................36, 38
Creating the 3D View............................ 58, 106 End Point for a Wavelength Series ...............39
Data Format................................................ 127 Enlarged Recordings.....................................28
Deleting Regions of Interest ....................... 122 Experiment ............................................45, 129
Detection ...................................................... 83 Experiment anlegen ....................................129
Detection Channel ........................................ 83 Experiment Legend .......................................48
Detection Pinhole ......................................... 29 Experiment Overview Viewer Window ........129
Detectors ...................................................... 22 Export ..........................................................116
Dichroic....................................................... 138 Exporting Quantification Data .....................116
Dichroic Filters.............................................. 22 File Type......................................................127
Diffraction Pattern......................................... 29 Film sequence .................................. 88, 89, 90
Digitalization ................................................. 47 First ...............................................................88
Displacement................................................ 44 First Button ....................................................88
Displaying an Image ..................................... 90 First Image of a Series ..................................88

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Fluorescence microscopy........................... 138 Image Tool Dialog Window,Basic Button 62, 71
Fluorite Objectives................................ 24, 138 Image Tool Dialog Window,Materials Button

..........................................................62, 131
Focal Plane................................................... 29
Image Tool Dialog Window,Multi Color Button
Focal shift compensation.............................. 48 ..................................................................62
Format Button............................................... 30 Immersion Objective .............................24, 138
Frame ........................................................... 28 In Focus.........................................................29
Gain Value .................................................... 25 Invariable Projection Axis .... 64, 71, 92, 95, 97,
100, 101, 102
Gallery .......................................................... 91
Isolines Image .............................................106
Gallery Button............................................... 91
Keyboard Shortcuts.......................................19
Gamma Curve .............................................. 77
Lambda Scan Button.....................................37
Glossary ..................................................... 138
Lambda Steps Button....................................41
Graphical Zoom ...................................... 52, 84
Last Button ....................................................89
Hardware Legend ......................................... 48
Last Image of a Series ..................................89
Hardware Settings .................................. 45, 48
LCS file formats...........................................144
Height ................................................. 134, 136
Legal notes......................................................5
Help .............................................................. 20
Legend ..........................................................48
Highpass............................................... 64, 131
Leica Confocal Software, LCS ......................13
Highpass Filter............................................ 131
Length of the measured section..................112
Histogram ................................................... 109
Leveling .................................................61, 141
Image Brightness.................................... 25, 77
Line..............................................................124
Image Contrast ....................................... 25, 77
line averaging ................................................45
Image Curvature Aberration ....................... 138
Line Button ..................................................124
Image Raster ................................................ 30
Linear Filters..........................................64, 131
Image Resolution.......................................... 47
Loading and Saving Parameter Settings ......22
Image Series 33, 36, 37, 40, 41, 88, 89, 90, 91
Long Pass Filter ..........................................138
Image Stack.................. 40, 41, 92, 95, 96, 105
Look-Up Table.........................................48, 84
Image Tool Button .................... 62, 64, 77, 131
Look-up Table Button ....................................84
Image Tool Dialog Window 62, 64, 71, 77, 131,
142 Lowpass Filter .......................................64, 131
Image Tool Dialog Window,3D Button ... 62, 64 LUT..........................................................48, 84
Image Tool Dialog Window,Amplitude Button Magnification ............................ 26, 51, 84, 108
........................................................... 62, 77
Materials Button ..........................................113

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Maximum Intensity...................................... 105 Number of Spatial Sections.....................36, 40
Maximum Projection 56, 64, 92, 95, 96, 97, 98, Number of Wavelength Steps .......................41
105
Numerical Aperture .......................................24
Maximum value .................... 77, 109, 110, 112
Nyquist Theorem ...........................................30
Measurement Slider ..................................... 53
Objective .......................................................24
Measuring ................................................... 112
Objective Button ............................................24
Measuring a Profile Along a Line Segment112,
134 Objective Dialog Window ..............................24
Measuring a Surface .................................. 113 Objective Nosepiece .....................................24
Measuring Roughness........................ 134, 136 Offset.............................................................44
Measuring Volumes.................................... 113 Offset Value...................................................25
Merging....................................................... 142 Open............................................................127
Merging Images............................................ 71 Open Button ................................................127
Minimum value ..................... 77, 109, 110, 112 Opening a File .............................................127
Mode Button ................................................. 31 Opening the Context-Sensitive Help .............20
Move Button ............................................... 108 Optimal Zoom Factors...................................26
Moving .................................................. 51, 108 Option New Window....................................129
Moving an Image in the Result Image........ 142 Options Dialog Window...............................129
Moving the 3D View.................................... 108 OR ...............................................................143
Moving the Region of Interest .................... 121 Original Image .............................................106
Moving the Region of Interest (ROI)... 120, 121 Orthogonal Projections...... 64, 71, 95, 97, 100,
101, 102
Multidimensional image data block .............. 41
Out of Focus..................................................29
Multiple Image .............................................. 87
Overlay ..........................................................57
Multiplication................................................. 77
Overlay Button...............................................87
Multiplying Images........................................ 77
Phase Button.................................................44
Neutral Filter ......................................... 22, 138
Phase Displacement .....................................44
New Experiment ......................................... 129
Phase Visualization .....................................138
New Experiment Button.............................. 129
Ping-pong Mode ............................................52
New Window................................................. 48
Pinhole Button ...............................................29
Next .............................................................. 88
Plane Objectives ...................................24, 138
Next Button ................................................... 88
Play/Stop Button............................................90
Next Image of a Series ................................. 88
Polygon .......................................................117

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Position of Maximum Value ........................ 110 ROI ....... 28, 116, 117, 118, 119, 120, 121, 122
Position of Minimum Value ......................... 110 Root mean square value ............ 109, 134, 136
Previous Button ............................................ 89 Rotate..........................................................121
Previous Image of a Series .......................... 89 Rotate Button ..............................................107
Printer Selection Dialog Window ................ 126 Rotating .......................................................107
Printing ............................................... 115, 126 Rotating the 3D View ..................................107
Printing Quantification Data........................ 115 Rotation Animation ........................................64
Processing Large Amounts of Data.............. 46 Save ............................................................127
Profile (z) Button......................................... 110 Save All .......................................................128
Profile Button .............................................. 112 Save All Button............................................128
Profile Peak ........................................ 134, 136 Save As .......................................................128
Profile Valley....................................... 134, 136 Save As Button............................................128
Progress Display .......................................... 58 Save Button.................................................127
Projection............................ 71, 92, 95, 96, 104 Saving .................................................127, 128
Projection Button .................................... 95, 96 Saving a File................................................127
Projection of an Image Stack ... 92, 95, 96, 104 Saving a File as...........................................128
Quantification........ 53, 112, 115, 116, 134, 136 Saving Measurement Points .......................134
Raw Data .................................................... 128 Saving the Viewer Window as a Template .129
Recording a Line Using the Averaging Method Scaling of the Measurement Curve...............53
................................................................. 45
Scan Field .....................................................44
Recording an Image Using the Averaging
Method ..................................................... 46 Scan Field Rotation Button ...........................44
Rectangle ........................................... 118, 124 Scan Format..................................................30
Rectangle Button ........................................ 124 Scan Mode ..............................................31, 58
Reducing ........................................ 51, 84, 108 Scan Speed...................................................32
Refraction Index ......................................... 138 Sections Button .............................................40
Region of Interest110, 116, 117, 118, 136, 138 Select ..........................................................120
Region of Interest as a Polygon ................. 117 Select LUT Dialog Window ...........................84
Region of Interest as a Rectangle .............. 118 Selecting a Scan Format...............................30
Region of Interest as an Ellipse.................. 116 Selecting a Scan Mode .................................31
Resolution..................................................... 30 Selecting a Scan Speed................................32
Resolution Power ......................................... 24 Selecting a unidirectional or bidirectional scan

..................................................................43

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Selecting an Image....................................... 90 Software Reference, Menu Functions...........16
Selecting an Objective.................................. 24 Spatial Image Series ...............................40, 41
Selecting Color Look-Up Tables....... 48, 83, 84 Spatial Representation ................................106
Selecting Color Look-Up Tables (LUT) ........ 84 Spectral Scan ..........................................37, 41
Selecting the Excitation Beam Splitter ......... 22 Spectral Series ........................................37, 41
Selecting the Excitation Wavelength ............ 22 Speed Button.................................................32
Selecting the Region of Interest ................. 120 speichern.....................................................127
Selection ................................................. 48, 90 Stack Processing of Data..............................46
Selection Button ........................................... 90 Standard deviation ..................... 109, 110, 112
Separating Images ....................................... 71 Start Point......................................................36
Serie ............................................................. 88 Start Point for a Spatial Series ................36, 37
Series.... 33, 36, 37, 40, 41, 88, 89, 90, 91, 105 Start Point for a Wavelength Series ..............37
Series Button ................................................ 36 Starting a Series Scan ..................................41
Series Image ................................................ 91 Starting and Ending a Film............................90
Series Scan Button....................................... 41 Statistical Calculations ................................109
Series Scan Overview Dialog Window ......... 36 Step Width.....................................................40
Setting Scan Parameters.............................. 35 Stereo View .............................................64, 71
Setting the Photomultiplier ........................... 25 Stokes Displacement ..................................138
Setting the z/y Position ................................. 33 Subtracting Images .......................................77
SFP Projection Image............... 56, 64, 92, 104 Subtraction ....................................................77
Sharp .................................................... 64, 131 Suppressing Noise ........................................46
Short Pass Filter ......................................... 138 Surface Structure ........................................105
Signal-to-noise Ratio .................................. 138 Taste First .....................................................88
Simulated Fluorescence Process (SFP) .... 104 Taste Image Tool ..................................77, 131
Simultaneous or Sequential Image Recording Taste Look-up Table .....................................84

................................................................. 22
Taste Snap ..................................................123
Single Image................................................. 86
Template .....................................................129
Single Scan .................................................. 34
Template Button ..........................................129
Skewness of the Distribution ...................... 109
Text Button ..................................................125
Smooth ................................................. 64, 131
Text Field.....................................................125
Snap ........................................................... 123
The arithmetical mean.........................110, 112
Snap Button................................................ 123
Thickness of the Optical Sections .................29

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Tiled .............................................................. 87 Viewer Options Dialog Window,Scan Progress

Icon ...........................................................58
Tiled Button .................................................. 87
Viewer Options Dialog Window,Surface
Time Button .................................................. 33 Calculation Icon ........................................61
Time Configuration Dialog Window .............. 33 Viewer Options Dialog Window,Surface View
Icon ...........................................................58
Time Series ............................................ 33, 41
Viewer Window........................................48, 84
Topographical Image............................ 61, 105
Viewing an Overlay Image ......................57, 87
Topography .......................................... 61, 105
Viewing Measurement Bar ............................52
Topography Button ..................................... 105
Viewing the Last Image of a Series...............89
Transparent Factor ........... 56, 64, 92, 102, 103
Viewing the Original Image .........................106
Transparent Projection 56, 64, 92, 95, 96, 102,
103 Viewing the Series Image .............................91
Triple Dichroic............................................. 138 Viewing z-Position .........................................52
Triple dichroic filters...................................... 22 Voxel ...........................................................138
Turning the Scan Field ................................. 44 Wavelength ...................................................41
Two-point measurement....... 53, 110, 113, 134 Wavelength Series ........................... 37, 39, 41
Undersampling ............................................. 30 Windows NT, Setting Up Users.....................12
Undo ........................................................... 106 Windows NT, starting ......................................7
Unidirectional................................................ 43 Wireframe....................................................106
Unidirectional Scan....................................... 43 Wizard .........................................................119
Unidirectional/ Bidirectional Scan Button ..... 43 Working Distance ..................................24, 138
UV-Lens-Wheel ............................................ 48 XOR.............................................................143
Variable Projection Axis..... 64, 92, 96, 98, 100, xy-Section......................................................40

101, 103
xy-Sections....................................................31
Variance ..................................... 109, 110, 112
xz-Section......................................................40
Viewer........................................................... 48
xz-Sections....................................................31
Viewer Options Dialog Window . 51, 52, 53, 56,
57, 58, 61 y Position.......................................................33
Viewer Options Dialog Window,3D Icon....... 51 Z Configuration Dialog Window.....................40
Viewer Options Dialog Window,Charts Icon. 53 z Position.......................................................33
Viewer Options Dialog Window,Display Icon 52 z/y Position ..............................................37, 38
Viewer Options Dialog Window,Overlay Icon57 z/y-Position Button ........................................33
Viewer Options Dialog Window,Projections Zoom .....................................................26, 108

Icon........................................................... 56
Zoom Button................................................108

Art.No.: 15-9330-033 / Vers.: 01082001
Index
Zoom In Button ............................................. 28 Zooming the 3D View..................................108

Art.No.: 15-9330-033 / Vers.: 01082001

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Leica Confocal Software

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REVISIONS AND CHANGES

User Manual Leica LCS english Page 5 of 164

performed entirely at the user's risk.

User Manual Leica LCS english Page 6 of 164

Starting up the system

If you miss-type the confirmation password, you will see a warning

User Manual Leica LCS english Page 7 of 164

The Windows NT Interface

User Manual Leica LCS english Page 8 of 164

Microsoft Mail (MS Mail) and Microsoft Exchange. If there is already an

Files that are overwritten due to an application using a duplicate filename

The Start Menu

User Manual Leica LCS english Page 9 of 164

Windows NT Workstation 4 does not actively track a link between an

Document files that are opened within an application (typically by

Setting the Time and Date

User Manual Leica LCS english Page 10 of 164

Depending on the shell configuration, systems connected to a network

Shut Down Windows NT

User Manual Leica LCS english Page 11 of 164

Setting Up Users Under WinNT

User Manual Leica LCS english Page 12 of 164

The Leica Confocal Software:

Starting the Software

User Manual Leica LCS english Page 13 of 164

The Experiment Software Concept

The Basic Structure of the User Interface

User Manual Leica LCS english Page 14 of 164

Opening Data Records

User Manual Leica LCS english Page 15 of 164

Data Organization by Grouping Experiments

Images that have been recorded in continuous scan mode are

User Manual Leica LCS english Page 16 of 164

The View Menu

The Macro Menu

User Manual Leica LCS english Page 17 of 164

The Tools Menu

The Window Menu

The Help Menu

User Manual Leica LCS english Page 18 of 164

LCS Software Functions

Contents of the Online Help

User Manual Leica LCS english Page 19 of 164

Keyword Search (Index)

Full-text Boolean Search

Opening the Context-Sensitive Help

! Click the Help button.

User Manual Leica LCS english Page 20 of 164

User Manual Leica LCS english Page 21 of 164

Setting the Beam Path

Selecting the excitation wavelength

Loading and Saving Parameter Settings

! Click the Save button.

! Select the Settings option from the Tools menu.

Click one of the user-defined or preset parameter configurations and then

User Manual Leica LCS english Page 22 of 164

Selecting the excitation beam splitter

! Neutral filters, such as the RT 30/70 filter, are used in reflection

Setting detectors and color look-up tables (LUT)

! Activate each of the desired detectors by clicking on its corresponding

! see Selecting Color Look-Up Tables